Detection and identification of enteric bacteria

ABSTRACT

The invention provides probes, antibodies and methods for detecting a gene that is only found in Enterobacteriaceae, the deoxyguanosine triphosphate triphosphohydrolase gene. These probes and methods are useful for the detecting whether test samples, including food and water samples, are infected with enteric bacteria.

FIELD OF THE INVENTION

[0001] The invention relates to detection of Enterobacteriaceae andidentification of the genus or genera of Enterobacteriaceae present in atest sample. In particular, the invention provides methods for detectingboth nucleic acids and the encoded enzyme (deoxyguanosine triphosphatetriphosphohydrolase) found only in Enterobacteriaceae. The nucleic acidsand methods provided by the invention are useful for determining whetherfood, water and other types of samples are contaminated with entericbacteria. Methods of the invention also permit identification of thetype of enteric bacteria present in a contaminated sample.

BACKGROUND OF THE INVENTION

[0002] Food poisoning and other food borne diseases that are caused byenteropathogenic bacteria account for millions of illnesses andthousands of deaths each year in the United States (1,2). The clinicalconditions that result from acute ingestion of pathogenic bacteriainclude diarrhea, vomiting, and dysentery (3). However, other moreserious medical complications may occur, such as renal and cardiacdisorders, neurological dysfunction, hemolytic uremia, and death (4).The situation in non-industrialized countries is even worse, where it isestimated that more than 10 percent of the population is chronicallyinflicted with food borne disease (5). Public health organizations havenot only been faced with an ever increasing rate of food poisoning casesin the United States, but with newly emerging bacterial food bornediseases (6, 7). In addition to human health issues, food borneillnesses take a continued and a heavy economic toll on society bylowering economic productivity and by stretching the available resourcesof local and national public health organizations (8).

[0003] The bacteria responsible for these human illnesses are from thetaxonomic family Enterobacteriaceae (9). The four main genera ofbacteria within this family that pose a risk to human health via foodborne illnesses are: Escherichia, Salmonella, Shigella, and Yersinia.All foodstuffs are susceptible to bacterial contamination of thesebacteria. The original sources of this contamination may be from animalhosts (for example, cows, chickens, or pigs) that harbor systemicinfections, from improper handling of otherwise uncontaminatedfoodstuffs (for example, poor worker hygiene), or from washingfoodstuffs in contaminated water.

[0004] Traditional food and restaurant inspection techniques have reliedupon visual inspection of foodstuffs and food preparation areas.However, foodstuffs contaminated with enteropathogenic bacteria oftenlook, smell and taste normal. Many of these pathogens may also survivethe cooking process (10, 11). When bacterial culturing is conducted,samples must be returned to a laboratory for microbiological testing.Such tests often take weeks to perform. Meanwhile, a potential healthrisk continues.

[0005] Studies by Quirk and Bessman (12) have revealed that a dGTPase isonly detected in bacteria belonging to the family Enterobacteriaceae.However, this reference does not provide nucleic acids probes andantibodies capable of detecting Enterobacteriaceae in general and alsodistinguishing between the various types of Enterobacteriaceae.

[0006] It is therefore imperative to develop faster and more reliabledetection methods that are sensitive and specific enough to identify notonly that enteropathogenic bacterial contamination exists in food andwater samples, but what type of enteropathogenic bacterial contaminationis present.

SUMMARY OF THE INVENTION

[0007] According to the invention, the enzyme deoxyguanosinetriphosphate triphosphohydrolase (dGTPase; E.C. 3.1.5.1) is found onlyin Enterobacteriaceae and detection of this enzyme is a specificindicator that Enterobacteriaceae pathogens are present in a testsample. The invention provides methods for identifying which genus orgenera of Enterobacteriaceae is present in a contaminated sample, aswell as antibodies and nucleic acids useful for detection ofEnterobacteriaceae dGTPase. Such methods may involve immunological,enzymatic, hybridization, nucleic acid amplification and relatedprocedures for detection and identification of Enterobacteriaceae.

[0008] The invention provides an isolated nucleic acid that includes SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18. Such nucleic acids can selectively hybridize toDNA from a bacteria of the family Enterobacteriaceae.

[0009] The invention also provides an isolated nucleic acid thatincludes SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, Thesenucleic acids can selectively hybridize to DNA from Escherichia colieven in the presence of DNA from at least one other bacterial species ofthe family Enterobacteriaceae such as Klebsiella, Salmonella, Shigellaor Yersinia.

[0010] The invention further provides an isolated nucleic acid thatincludes SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.These nucleic acids can selectively hybridize to DNA from Salmonellatyphymurium, in the presence of DNA from Klebsiella or Escherichia.

[0011] The invention also provides an isolated nucleic acid thatincludes SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.These nucleic acids can selectively hybridize to DNA from Klebsiellaoxytoca, in the presence of DNA from Salmonella or Escherichia.

[0012] The invention further provides a biosensor chip that includes asolid support and a nucleic acid including SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0013] The invention also provides a method of detecting the presence ofenteric bacteria in a test sample that includes contacting the testsample with probe under stringent hybridizations conditions, anddetecting hybridization between the probe and a nucleic acid in the testsample. Such a probe can include a nucleic acid that includes, forexample, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, or SEQ ID NO:18. Such enteric bacteria are of the familyEnterobacteriaceae. This method may further include DNA amplification,for example, polymerase chain reaction.

[0014] The invention further provides a method of detecting the presenceof any species of enteric bacteria in a test sample that includescontacting the test sample with probe under stringent hybridizationsconditions, and detecting hybridization between the probe and a nucleicacid in the test sample. Probes useful in this method include nucleicacids with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6. This method may further include DNA amplification, for example,polymerase chain reaction.

[0015] The invention also provides a method of detecting the presence ofEscherichia in a test sample that includes contacting the test samplewith probe under stringent hybridizations conditions, and detectinghybridization between the probe and a nucleic acid in the test sample.Such a probe may be an isolated nucleic acid that includes SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. These probes can selectivelyhybridize to DNA from Escherichia coli in the presence of DNA fromKlebsiella, Salmonella, Shigella or Yersinia. This method may furtherinclude DNA amplification, for example, polymerase chain reaction.

[0016] The invention further provides a method of detecting the presenceof Salmonella in a test sample that includes contacting the test samplewith probe under stringent hybridizations conditions, and detectinghybridization between the probe and a nucleic acid in the test sample.Such a probe may be an isolated nucleic acid that includes SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. These probes canselectively hybridize to DNA from Salmonella typhymurium in the presenceof DNA from Klebsiella or Escherichia. This method may further includeDNA amplification, for example, polymerase chain reaction.

[0017] The invention also provides a method of detecting the presence ofKlebsiella in a test sample that includes contacting the test samplewith probe under stringent hybridizations conditions, and detectinghybridization between the probe and a nucleic acid in the test sample.Such a probe may be an isolated nucleic acid that includes SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. These probes canselectively hybridize to DNA from Klebsiella oxytoca in the presence ofDNA from Salmonella or Escherichia. This method may further include DNAamplification, for example, polymerase chain reaction.

[0018] The invention also provides method for detecting enteric bacteriain a test sample that includes contacting a test sample with a biosensorchip that has a solid support and an antibody that can bind to dGTPasefrom Enterobacteriaceae, and detecting whether dGTPase is bound to thebiosensor chip. Biosensor chips that include such a solid support and anantibody that can selectively bind to dGTPase from Enterobacteriaceaeare also provided by the invention.

[0019] Antibodies that can bind to dGTPase from Enterobacteriaceae,include, for example, antibodies directed against a peptide having SEQID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, or SEQ ID NO:36.

DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 illustrates the loss of activity in enzyme preparationsfrom several species of Enterobacteriaceae as a function of increasingamounts of anti-dGTPase polyclonal antibody (pAb). The anti-dGTPasepolyclonal antibody was raised against dGTPase isolated from E. coli.Inhibition of dGTPase isolated from Y. enterocolitica (open circles), E.aerogens (closed triangles), P. vulgaris (open diamonds) K. oxytoca(open triangles), S. typhimurium (open squares), S. boydii (closedcircles), and C. davisae (closed squares) by the anti-dGTPase pAb isshown.

[0021]FIG. 2 illustrates the loss of activity in enzyme preparationsfrom several species of Enterobacteriaceae as a function of increasingamounts of anti-dGTPase polyclonal antibody (pAb). The anti-dGTPasepolyclonal antibody was raised against dGTPase isolated from S.marcescens. Inhibition of dGTPase isolated from S. typhimurium (opensquares), K. oxytoca (open triangles), S. boydii (closed circles), P.vulgaris (open diamonds), Y. enterocolitica (open circles), H. alvei(closed diamonds), E. aerogens (closed triangles), and S. marcescens(closed squares) by the anti-dGTPase pAb is shown.

[0022]FIG. 3 provides results of an ELISA analysis of cross reactivitybetween anti-E. coli dGTPase pAb and 1 mg of an adsorbed Fraction IIIenzyme preparation from: E. coli 0157 (closed circles), S. boydii (opencircles), S. typhimurium (closed squares), K. oxytoca (closedtriangles), E. aerogens (closed diamonds), C. davisae (open squares), Y.enterocolitica (open diamonds), and C. freundii (open triangles).

[0023]FIG. 4 provides results of an ELISA analysis of cross reactivitybetween anti S. marcescens dGTPase pAb and 1 mg of adsorbed Fraction IIIenzyme preparation from: S. typhimurium (closed triangles), E. coli(open triangles), H. alvei (open squares), E. aerogens (closed circles),Y. enterocolitica (open circles), and P. vulgaris (closed squares).

[0024]FIG. 5A provides an SDS PAGE analysis of enteric dGTPases fromseveral species isolated using an immunoaffinity column chromatographycolumn of anti-Escherichia dGTPase pAb (lanes 2-5) or anti-SerratiadGTPase pAb (lanes 6-9). Lane 1, molecular weight markers (65 kDa, 45kDa, 24 kDa from top to bottom); Lane 2, Salmonella; Lane 3, Shigella;Lane 4, Klebsiella; Lane 5, Cedecca; Lane 6, Yersinia; Lane 7, Proteus;Lane 8, Enterobacter; Lane 9, Hafnia.

[0025]FIG. 5B provides a Western blot analysis using antibodies raisedagainst two different isolates of dGTPase. Anti-E. coli dGTPase pAb wasused for detection of dGTPase in crude S. marcescens protein extract(lane 1) and in crude E. coli protein extract (lane2). Anti-S.marcescens dGTPase pAb was used for detection of dGTPase in crude S.marcescens protein extract (lane 3) and in crude E. coli protein extract(lane 4).

[0026]FIG. 6 provides an surface plasmon resonance (SPR) sensogramshowing the reactivity of tethered pAbs against dGTPase from variousbacteria. Relative SPR signal intensity is plotted as a function of timefor anti-E. coli dGTPase pAbs reacted with crude bacterial extract of E.coli (Ec), S. boydii (Sb), C. daviseae (Cd), K. oxytoca (Ko), S.typhimurium (St), C. freundii (Cf), E. aerogens (Ea), and S. aureus (Sa)(non enteric bacterial control). Flow rate over the sensor surface was50 μL per minute.

[0027]FIG. 7 provides an SPR sensogram showing the reactivity oftethered pAbs-against dGTPase from various bacteria. Relative SPR signalintensity is plotted as a function of time for: anti-S. marcescensdGTPase pAbs reacted with crude bacterial extract of S. marcescens (Sm),E. aerogens (Ea), Y. enterocolitica (Ye), P. vulgaris (Pv), E. coli(Ec), and S. typhimurium (St). Flow rate over the sensor surface was 50μL per minute.

[0028]FIG. 8 provides a curve illustrating the titration of the SPRbiosensors with purified dGTPase. The curves represent the relative SPRsignal intensity for a given amount of enzyme reacted with the surfaceat 400 seconds (after binding is complete, see FIG. 6). In betweensample points, the chip was washed with 2 M potassium thiocyanate inorder to remove bound dGTPase, followed by a 10 minute buffer wash,followed by the next dGTPase concentration. The anti E. coli pAb surface(open circles) was reacted with E. coli dGTPase, and the anti S.marcescens pAb surface (closed circles) was reacted with S. marcescensdGTPase. The abscissa scale runs from 0 to 100 ng.

[0029]FIG. 9 provides the DNA sequence of the E. coli dgt gene.

[0030]FIG. 10 illustrates that the methods of the invention can detectas few as 100 Enterobacteriaceae (in this case E. coli) in a testsample. Primer set 1 was used to amplify the 184 bp fragment fromapproximately 1000 E. coli bacteria (lane 1), 100 bacteria (lane 2), and10 bacteria (lane3). Thirty cycles of the PCR reaction were employed.The band in lane 3 is just visible in the original gel. The gel is 1.2%agarose and is stained with ethidium bromide.

[0031]FIG. 11 illustrates the specificity of the nucleic acid probesprovided by the invention. In this experiment, DNA was obtained fromapproximately 200 cfu from various species of Enterobacteriaceae. TheseDNA isolates were subjected to PCR amplification using primer set 5 thatspecifically amplify only E. coli dgt nucleic acids to produce a 213 bpfragment. The products of amplification were separated on a 1.2% agarosegel and stained with ethidium bromide. Each lane contains theamplification products from a separate genus of substrate DNA: Lane1—Klebsiella; Lane 2—Salmonella; Lane 3—Shigella; Lane 4—Yersinia; Lane5—Escherichia. The 213 bp fragment is correctly amplified only in the E.coli lane.

[0032]FIG. 12 further illustrates the specificity of the nucleic acidprobes provided by the invention under conditions where different typesof bacteria are present in the test sample and may compete for primerbinding and amplification. In this experiment, DNA was obtained fromapproximately 200 cfu of two different genera of Enterobacteriaceae andmixed together. After amplification, the products were separated on a1.2% agarose gel and stained with ethidium bromide. The species ofbacterial DNA present in the samples and the primers used to test thespecificity of amplification are provided below. Lane Bacteria PrimerPair Band size (bp) 1 Escherichia + Klebsiella 7 and 11 82(Escherichia) + 213 (Klebsiella) 2 Escherichia + Salmonella 7 and 8 82(Escherichia) + 213 (Salmonella) 3 Salmonella + Klebsiella 8 and 13 213(Salmonella) + 82 (Klebsiella) 4 Klebsiella + Escherichia 6 251(Escherichia) 5 Salmonella + Escherichia 6 251 (Escherichia)

[0033] Primer pairs 5, 6 and 7 were designed to be specific forEscherichia. Primer pairs 8, 9 and 10 were designed to be specific forSalmonella. Primer pairs 11, 12 and 13 were designed to be specific forKlebsiella. Each of the primer pairs employed actually synthesized a DNAfragment of the predicted size for a particular genus of bacteria. Thus,the methods of the invention discriminate between DNA from relatedEnterobacteriaceae and correctly identify which bacterial genus ispresent in a mixed bacterial culture.

[0034]FIG. 13 illustrates that the methods of the invention can detectand identify Enterobacteriaceae within an actual meat sample (chicken).Ten μL of chicken fluid (blood) was used in a PCR reaction with thefollowing primer sets: Lane Primer Bacterial Type Primer Observed bandsNo. Set Designed to Detect (Same as Predicted) 1 2 Any enteric bacteria213 2 5 Escherichia only 213 3 11 Klebsiella only None 4 8 Salmonellaonly 213 5 8 (2×) Salmonella only 213 6 5 + 7 Escherichia only 251 + 837 1 Any enteric bacteria 213

[0035] Twice the amount of primer set 8 was used in the reaction forlane 5 and for lane 4. The bands in lanes 6 and 7 were faint but werevisibly present. These results indicate that the chicken sample wascontaminated with both Salmonella and Escherichia, but not withKlebsiella.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Deoxyguanosine triphosphate triphosphohydrolase (dGTPase) is theonly enzyme that has been identified as being specifically localized inEnterobacteriaceae. No dGTPase activity is detected in extracts ofeukaryotic cells, including extracts of rat and chicken livers, chickenand Drosophila embryos, and yeast cells. The enzyme is expressed in allof the pathogenicly important genera of the Enterobacteriaceae, but notin the Erwinia species. Surprisingly, the enzyme is also not expressedin the closely related Vibrio genus. Moreover, the dGTPase polypeptideshows little interspecies variation in expression level. This narrowtaxonomic distribution of dGTPase may be utilized to identifyenteropathogenic bacteria in all sorts of test samples.

[0037] Deoxyguanosine triphosphate triphosphohydrolase was discovered byKornberg et al. (15) as a contaminant during the purification of DNApolymerase I. The enzyme was partially purified and found to catalyzethe hydrolysis of deoxyguanosine triphosphate to deoxyguanosine andinorganic tripolyphosphate:

dGTP→

dGuo+PPPi

[0038] This reaction is unique among all known nucleosidetriphosphatases, which either form ortho or pyrophosphate as endproducts. To date, dGTPase is the only known triphosphohydrolase. ThedGTPase enzyme is a thermostable tetrameric protein (13, 14) and it isimmunogenic (12). The enzymological and structural properties of dGTPasefrom the other enteric bacteria (16) are similar to dGTPase isolatedfrom Escherichia.

[0039] Definitions

[0040] The term “amino acid sequence” refers to the positionalarrangement and identity of amino acids in a peptide, polypeptide orprotein molecule. Use of the term “amino acid sequence” is not meant tolimit the amino acid sequence to the complete, native amino acidsequence of a peptide, polypeptide or protein.

[0041] The term “coding region” refers to the nucleotide sequence thatcodes for a protein of interest or to a functional RNA of interest, forexample antisense RNA or a nontranslated RNA. The coding region of aprotein is bounded on the 5′ side by the nucleotide triplet “ATG” thatencodes the initiator methionine and on the 3′ side by one of the threetriplets that specify stop codons (i.e., TAA, TAG, TGA). The codingregion may be present in either a cDNA, genomic DNA or RNA form.

[0042] “Complementary” or “complementarity” are used to define thedegree of base-pairing or hybridization between nucleic acids. Forexample, as is known to one of skill in the art, adenine (A) can formhydrogen bonds or base pair with thymine (T) and guanine (G) can formhydrogen bonds or base pair with cytosine (C). Hence, A is complementaryto T and G is complementary to C. Complementarity may be complete whenall bases in a double-stranded nucleic acid are base paired.Alternatively, complementarity may be “partial,” in which only some ofthe bases in a nucleic acid are matched according to the base pairingrules. The degree of complementarity between nucleic acid strands has aneffect on the efficiency and strength of hybridization between nucleicacid strands.

[0043] “Control sequences” or “regulatory sequences” are DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. Control sequences that are suitable forprokaryotic cells include, for example, a promoter, and optionally anoperator sequence, and a ribosome binding site.

[0044] “Expression” refers to the transcription and/or translation of anendogenous or exogeneous gene in an organism. Expression generallyrefers to the transcription and stable accumulation of mRNA. Expressionmay also refer to the production of protein.

[0045] The term “gene” is used broadly to refer to any segment ofnucleic acid associated with a biological function. The term “gene”encompasses the coding region of a protein, polypeptide, peptide orstructural RNA. The term “gene”also includes sequences up to a distanceof about 2 kb on either end of a coding region. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain control or regulatorysequences such as promoters and enhancers or other recognition orbinding sequences for proteins that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation as well as recognition sequences for otherproteins. A protein or polypeptide encoded in a gene may be full lengthor any portion thereof, so that all activities or functional propertiesare retained, or so that only selected activities (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthprotein or polypeptide are retained. The protein or polypeptide mayinclude any sequences necessary for the production of a proprotein orprecursor polypeptide. The term “gene” encompasses both cDNA and genomicforms of a coding region. A genomic form of a coding region may beinterrupted with non-coding sequences termed “introns.” The term “nativegene” refers to gene that is naturally present in the genome of anuntransformed cell.

[0046] “Genome” refers to the complete genetic material that isnaturally present in an organism and is transmitted from one generationto the next.

[0047] The terms “heterologous nucleic acid,” or “exogenous nucleicacid” refer to a nucleic acid that originates from a source foreign tothe particular host cell or, if from the same source, is modified fromits original form. Thus, a heterologous gene in a host cell includes agene that is endogenous to the particular host cell but has beenmodified through, for example, the use of DNA shuffling. The terms alsoinclude non-naturally occurring multiple copies of a naturally occurringnucleic acid. Thus, the terms refer to a nucleic acid segment that isforeign or heterologous to the cell, or normally found within the cellbut in a position within the cell or genome where it is not ordinarilyfound.

[0048] The term “homology” refers to a degree of similarity between anucleic acid and a reference nucleic acid or between a polypeptide and areference polypeptide. Homology may be partial or complete. Completehomology indicates that the nucleic acid or amino acid sequences areidentical. A partially homologous nucleic acid or amino acid sequence isone that is not identical to the reference nucleic acid or amino acidsequence. Hence, a partially homologous nucleic acid has one or morenucleotide differences in its sequence relative to the nucleic acid towhich it is being compared. The degree of homology may be determined bysequence comparison. Alternatively, as is well understood by thoseskilled in the art, DNA-DNA or DNA-RNA hybridization, under varioushybridization conditions, may provide an estimate of the degree ofhomology between nucleic acids, (see, e.g., Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.).

[0049] “Hybridization” refers to the process of annealing complementarynucleic acid strands by forming hydrogen bonds between nucleotide baseson the complementary nucleic acid strands. Hybridization, and thestrength of the association between the nucleic acids, is impacted bysuch factors as the degree of complementary between the hybridizingnucleic acids, the stringency of the conditions involved, the T_(m) ofthe formed hybrid, the length of the hybridizing nucleic acids and theG:C ratio of those nucleic acids.

[0050] An “initiation site” is region surrounding the position of thefirst nucleotide that is part of the transcribed sequence, which isdefined as position +1. All nucleotide positions of the gene arenumbered by reference to the first nucleotide of the transcribedsequence, which resides within the initiation site. Downstream sequences(i.e. sequences in the 3′ direction) are denominated positive, whileupstream sequences (i.e. sequences in the 5′ direction) are denominatednegative.

[0051] An “isolated” or “purified” nucleic acid or an “isolated” or“purified” polypeptide is a nucleic acid or polypeptide that, by thehand of man, exists apart from its native environment and is thereforenot a product of nature. An isolated nucleic acid or polypeptide mayexist in a partially purified or substantially purified form. Anisolated nucleic acid or polypeptide may also exist in a non-nativeenvironment such as, for example, a transgenic host cell.

[0052] The term “label” refers to any atom or molecule that may be usedto provide a detectable (preferably quantifiable) signal, and that maybe attached to a nucleic acid or protein. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, and thelike.

[0053] The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as thereference sequence explicitly indicated.

[0054] The term “oligonucleotide” as used herein is defined as amolecule comprised of two or more deoxyribonucleotides orribonucleotides, desirably more than three, and usually more than ten.There is no precise upper limit on the size of an oligonucleotide.However, in general, an oligonucleotide is shorter than about 250nucleotides, more desired oligonucleotides are shorter than about 200nucleotides and even more desired oligonucleotides are shorter thanabout 100 nucleotides. Most desired oligonucleotides are shorter thanabout 50 nucleotides. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

[0055] The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence.

[0056] The terms “initiation codon” and “termination codon” refer to aunit of three adjacent nucleotides (‘codon’) in a coding sequence thatspecifies initiation and chain termination, respectively, of proteinsynthesis (mRNA translation).

[0057] “Operably linked” means that two or more nucleic acids are placedin a functional relationship with each other. For example, nucleic acidsencoding a presequence or secretory leader may be operably linked tonucleic acids encoding a polypeptide, and expressed as a pre-proteinthat participates in the secretion of the protein; a promoter orenhancer may be operably linked to a coding sequence and effect thetranscription of the sequence; or a ribosome binding site may beoperably linked to a coding sequence and positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous and, in the case of an encoded polypeptidesequence, in reading phase and generally contiguous. However, enhancersdo not have to be contiguous.

[0058] The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein

[0059] As used herein the term “stringency” is used to define theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acids that have a highfrequency of complementary base sequences. With “weak” or “low”stringency conditions nucleic acids the frequency of complementarysequences is usually less, so that nucleic acids with differingsequences may be detected and/or isolated.

[0060] The terms “substantially similar” and “substantially homologous”refer to nucleotide and amino acid sequences that represent functionalequivalents of the instant inventive sequences. For example, alterednucleotide sequences that simply reflect the degeneracy of the geneticcode but nonetheless encode amino acid sequences that are identical tothe inventive amino acid sequences are substantially similar to theinventive sequences. In addition, amino acid sequences that aresubstantially similar to the instant sequences are those wherein overallamino acid identity is sufficient to provide an active, thermally stabledGTPase. For example, amino acid sequences that are substantiallysimilar to the sequences of the invention are those wherein the overallamino acid identity is 80% or greater, desirably 90% or greater, andmore desirably 95% or greater relative to the amino acid sequences ofthe invention.

[0061] By “thermostable” is meant that the enzyme remains has an optimaltemperature of activity at a temperature greater than about 37° C. to42° C. Desired polymerase enzymes have an optimal temperature foractivity of between about 50° C. and 75° C., more desired enzymes havean optimal temperature between 55° C. and 70° C., and most desiredenzymes have an optimal temperature between 60° C. and 65° C.

[0062] The “variant”of a nucleic acid, protein, polypeptide or peptide,means that the variant nucleic acid, protein, polypeptide or peptide hasa related but different sequence than a reference nucleic acid, protein,polypeptide or peptide, respectively. A variant nucleic acid differs innucleotide sequence from a reference nucleic acid whereas a variantnucleic acid, protein, polypeptide or peptide differs in amino acidsequence from the reference nucleic acid, protein, polypeptide orpeptide, respectively. A variant and reference nucleic acid, protein,polypeptide or peptide may differ in sequence by one or moresubstitutions, insertions, additions, deletions, fusions andtruncations, which may be present in any combination. Differences may beminor (e.g. a difference of one nucleotide or amino acid) or moresubstantial. However, the sequence of the variant is not so differentfrom the reference that one of skill in the art would not recognize thatthe variant and reference are related in structure and/or function.Generally, differences are limited so that the reference and the variantare closely similar overall and, in many regions, identical.

[0063] The term “vector” is used to refer to a nucleic acid that cantransfer nucleic acid segment(s) from one cell to another. A “vector”includes, inter alia, any plasmid, cosmid, phage or nucleic acid indouble or single stranded linear or circular form that may or may not beself transmissible or mobilizable, and that can transform prokaryotic oreukaryotic host either by integration into the cellular genome or byexisting extrachromosomally (e.g. autonomous replicating plasmid with anorigin of replication). Vectors used in bacterial systems often containan origin of replication so that the vector may replicate independentlyof the bacterial chromosome. The term “expression vector” refers to avector containing an expression cassette.

[0064] The term “wild-type” refers to a gene or gene product that hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is the gene that is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product.Naturally-occurring mutants may be isolated; these are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product.

[0065] Enterobacteriaceae dGTPase Nucleic Acids

[0066] The invention is directed to nucleic acids encoding adeoxyguanosine triphosphate triphosphohydrolase enzyme from any speciesof the family Enterobacteriaceae. Variant nucleic acids that encode anactive deoxyguanosine triphosphate triphosphohydrolase enzyme are alsocontemplated by the invention. Any fragment of a nucleic acid is alsoencompassed by the invention so long as it can specifically hybridize toa nucleic acid encoding a deoxyguanosine triphosphatetriphosphohydrolase enzyme from any species of the familyEnterobacteriaceae. The nucleic acids of the invention are isolated orsubstantially purified nucleic acids. In particular, the isolatednucleic acids of the invention are free of nucleic acids that encodeproteins and that naturally flank the present nucleic acids in thegenomic DNA of the organism from which the nucleic acid is derived.Nucleic acids of the invention may be used to detect any bacterialspecies of the family Enterobacteriaceae. Detection may be by anyprocedure known to one of skill in the art, for example, any availablehybridization, amplification or related procedure.

[0067] For example, the invention is directed to nucleic acids encodingall or portions of a deoxyguanosine triphosphate triphosphohydrolaseenzyme from the genus Cedecca, Citrobacter, Enterobacter, Escherichia,Hafnia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, orYersinia. In one embodiment, the nucleic acid is all or a part of thenucleic acid encoding Escherichia coli deoxyguanosine triphosphatetriphosphohydrolase, that has the following sequence (SEQ ID NO:1):ATGGCACAGATTGATTTCCGAAAAAAAATAAACTGGCATCGTCGTTACC GTTCACCGCAGGGCGTTAAAACCGAACATGAGATCCTGCGGATCTTCGAGAG CGATCGCGGGCGTATCATCAACTCTCCGGCAATTCGTCGTCTGCAACAAAA GACCCAGGTTTTTCCACTGGAGCGCAATGCCGCCGTGCGCACGCGTCTTACC CACTCGATGGAAGTCCAGCAGGTGGGGCGCTACATCGCCAAAGAAATTTTA AGCCGTCTGAAAGAGCTTAAATTACTGGAAGCATACGGCCTGGATGAACTG ACCGGTCCCTTTGAAAGCATTGTTGAGATGTCATGCCTGATGCACGATATCGG CAATCCGCCGTTTGGTCATTTTGGCGAAGCGGCGATAAATGACTGGTTTCGCC AACGTTTGCACCCGGAAGATGCCGAAAGCCAGCCTCTGACTGACGATCGCTG CAGCGTGGCGGCACTACGTTTACGGGACGGGGAAGAACCGCTTAACGAGCTG CGGCGCAAGATTCGTCAGGACTTATGTCATTTTGAGGGGAATGCACAAGGCA TTCGCCTGGTGCATACATTGATGCGGATGAATCTCACCTGGGCACAGGTTGG CGGTATTTTAAAATATACCCGTCCGGCGTGGTGGCGTGGCGAAACGCCTGAG ACACATCACTATTTAATGAAAAAGCCGGGTTATTATCTTTCTGAAGAAGCCTA TATTGCCCGGTTGCGTAAAGAACTTAATTTGGCGCTTTACAGTCGTTTTCCA TTAACGTGGATTATGGAAGCTGCCGACGACATCTCCTATTGTGTGGCAGACC TTGAAGATGCGGTAGAGAAAAGAATATTTACCGTTGAGCAGCTTTATCATCA TTTGCACGAAGCGTGGGGCCAGCATGAGAAAGGTTCGCTCTTTTCGCTGGTG GTTGAAAATGCCTGGGAAAAATCACGCTCAAATAGTTTAAGCCGCAGTACGG AAGATCAGTTTTTTATGTATTTACGGGTAAACACCCTAAATAAACTGGTACC CTACGCGGCACAACGATTTATTGATAATCTGCCTGCGATTTTCGCCGGAACGT TTAATCATGCATTATTGGAAGATGCCAGCGAATGCAGCGATCTTCTTAAGCT ATATAAAAATGTCGCTGTAAAACATGTGTTTAGCCATCCAGATGTCGAGCGG CTTGAATTGCAGGGCTATCGGGTCATTAGCGGATTATTAGAGATTTATCGTCC TTTATTAAGCCTGTCGTTATCAGACTTTACTGAACTGGTAGAAAAAGAACG GGTGAAACGTTTCCCTATTGAATCGCGCTTATTCCACAAACTCTCGACGCCGC ATCGGCTGGCCTATGTCGAGGCTGTCAGTAAATTACCGTCAGATTCTCCTGA GTTTCCGCTATGGGAATATTATTACCGTTGCCGCCTGCTGCAGGATTATATC AGCGGTATGACCGACCTCTATGCGTGGGATGAATACCGACGTCTGATGGCC GTA GAACAATAA

[0068] Fragments and variant nucleic acids of the deoxyguanosinetriphosphate triphosphohydrolase nucleic acids provided herein are alsoencompassed by the invention. Nucleic acid “fragments” encompassed bythe invention are of two general types. First, fragment nucleic acidsthat do not encode a full length dGTPase but do encode a polypeptidewith dGTPase activity are within the scope of the invention. Second,fragments of a nucleic acids identified herein that are useful ashybridization probes or primers but generally do not encode dGTPaseactivity are also included in the invention.

[0069] Fragments may be obtained or developed using the E. coli dGTPasenucleic acid sequence (SEQ ID NO:1), or dGTPase nucleic acid sequencesfrom other enteric species of the family Enterobacteriaceae. OtherdGTPase nucleic acid sequences may be found in the Genebank sequencerepositories. Fragments used as primers may be designed to amplify anyenteric dGTPase nucleic acid when the primer sequence is selected froman areas of sequence identity or conservation between enteric species.On the other hand, primer sequences may be designed to be selective fora specific genus or species of Enterobacteriaceae by selecting asequence that is unique to that genus or species. One of skill in theart can readily design such primer sequences.

[0070] Thus, fragments of the invention may range from at least about 9nucleotides, about 12 nucleotides, about 15 nucleotides, about 17nucleotides, about 18 nucleotides, about 20 nucleotides, about 50nucleotides, about 100 nucleotides or more. In general, a fragmentnucleic acid of the invention can have any upper size limit so long asit is related in sequence to the nucleic acids of the invention, but isnot full length.

[0071] Nucleic acid fragments of the invention can be used, for example,as hybridization probes for detecting or identifying dGTPase nucleicacids or as primers for DNA synthesis, DNA sequencing or DNAamplification of dGTPase nucleic acids. Many fragments can be obtainedfrom SEQ ID NO:1. Examples of nucleic acid fragments of the inventioninclude any of SEQ ID NO:1-18.

[0072] By “variants” is intended substantially similar or substantiallyhomologous sequences. For nucleotide sequences, variants include thosesequences that, because of the degeneracy of the genetic code, encodethe identical amino acid sequence of a native dGTPase protein. Naturallyoccurring allelic variants include any Enterobacteriaceae dGTPasenucleic acid not specifically listed herein and are encompassed withinthe invention. Variant nucleic acids may be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques. Variantnucleic acids also include those that encode polypeptides that do nothave amino acid sequences identical to that of a native dGTPase protein,but that still encode an active dGTPase.

[0073] As is known by one of skill in the art, the genetic code is“degenerate,” meaning that several trinucleotide codons can encode thesame amino acid. This degeneracy is apparent from Table 1. TABLE 1Second Position 1st Position T C A G 3rd Position T TTT = Phe TCT = SerTAT = Tyr TGT = Cys T T TTC = Phe TCC = Ser TAC = Tyr TGC = Cys C T TTA= Leu TCA = Ser TAA = Stop TGA = Stop A T TTG = Leu TCG = Ser TAG = StopTGG = Trp G C CTT = Leu CCT = Pro CAT = His CGT = Arg T C CTC = Leu CCC= Pro CAC = His CGC = Arg C C CTA = Leu CCA = Pro CAA = Gln CGA = Arg AC CTG = Leu CCG = Pro CAG = Gln CGG = Arg G A ATT = Ile ACT = Thr AAT =Asn AGT = Ser T A ATC = Ile ACC = Thr AAC = Asn AGC = Ser C A ATA = IleACA = Thr AAA = Lys AGA = Arg A A ATG = Met ACG = Thr AAG = Lys AGG =Arg G G GTT = Val GCT = Ala GAT = Asp GGT = Gly T G GTC = Val GCC = AlaGAC = Asp GGC = Gly C G GTA = Val GCA = Ala GAA = Gln GGA = Gly A G GTG= Val GCG = Ala GAG = Gln GGG = Gly G

[0074] Hence, many changes in the nucleotide sequence of the variant maybe silent and may not alter the amino acid sequence encoded by thenucleic acid. Where nucleic acid sequence alterations are silent, avariant nucleic acid will encode a polypeptide with the same amino acidsequence as the reference nucleic acid.

[0075] Nucleic acid sequences of the invention also encompass variantswith degenerate codon substitutions, and complementary sequencesthereof, as well as the sequence explicitly specified by a SEQ ID NO.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the reference codon is replaced by any ofthe codons for the amino acid specified by the reference codon. Ingeneral, the third position of one or more selected codons can besubstituted with mixed-base and/or deoxyinosine residues as disclosed byBatzer et al., Nucleic Acid Res., 19, 5081 (1991) and/or Ohtsuka et al.,J. Biol. Chem., 260, 2605 (1985); Rossolini et al., Mol. Cell. Probes,8, 91 (1994).

[0076] However, the invention is not limited to silent changes in thepresent nucleotide sequences but also includes variant nucleic acidsequences that alter the amino acid sequence of a polypeptide of theinvention. According to the present invention, variant and referencenucleic acids of the invention may differ in the encoded amino acidsequence by one or more substitutions, additions, insertions, deletions,fusions and truncations, which may be present in any combination, solong as an active, dGTPase is encoded by the variant nucleic acid. Suchvariant nucleic acids will not encode exactly the same amino acidsequence as the reference nucleic acid, and are described herein ashaving “non-silent” sequence changes.

[0077] Variant nucleic acids with silent and non-silent changes can bedefined and characterized by the degree of homology to the referencenucleic acid. Desired variant nucleic acids are “substantiallyhomologous” to the reference nucleic acids of the invention. Asrecognized by one of skill in the art, such substantially similarnucleic acids can hybridize under stringent conditions with thereference nucleic acids identified by SEQ ID Nos herein. These types ofsubstantially homologous nucleic acids are encompassed by thisinvention.

[0078] Generally, nucleic acid variants of the invention will have atleast 50, 60, to 70% sequence identity to the reference nucleotidesequence defined herein. Desired nucleic acid variants of the inventionwill have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,sequence identity to the reference nucleotide sequence defined herein.More desired nucleic acid variants of the invention will have at least80%, 81%, 82%, 83% or 84% sequence identity to the reference nucleotidesequence defined herein. Even more desired nucleic acids of theinvention will have at least at least 85%, 86%, 87%, 88% or 89% sequenceidentity to the reference nucleotide sequence defined herein. Even moredesired nucleic acids of the invention will have at least 90%, 91%, 92%,93% or 94% sequence identity to the reference nucleotide sequencedefined herein. Most desired nucleic acids of the invention will have atleast at least 95%, 96%, 97%, to 98% sequence identity to the referencenucleotide sequence defined herein.

[0079] Variant nucleic acids can be detected and isolated by standardhybridization procedures.

[0080] Hybridization to detect or isolate such sequences is generallycarried out under stringent conditions. “Stringent hybridizationconditions” and “stringent hybridization wash conditions” in the contextof nucleic acid hybridization experiments such as Southern and Northernhybridization are sequence dependent, and are different under differentenvironmental parameters. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Laboratory Techniques in Biochemistry andMolecular biology-Hybridization with Nucleic Acid Probes, page 1,chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” Elsevier, New York (1993). See also, J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, N.Y., pp 9.31-9.58 [1989].

[0081] The invention also provides methods for detection and isolationof variant nucleic acids encoding dGTPase activity. The methods involvehybridizing at least a portion of a nucleic acid comprising SEQ ID NOS:1to 14 to a sample nucleic acid, thereby forming a hybridization complex;and detecting the hybridization complex. The presence of the complexcorrelates with the presence of a variant dGTPase nucleic acid. Ingeneral, the portion of a nucleic acid used for hybridization is atleast fifteen nucleotides long. Examples of nucleic acids used inhybridization methods include nucleic acids comprising SEQ ID NOS:1-18.Hybridization is performed under hybridization conditions that aresufficiently stringent to permit detection and isolation of homologousnucleic acids.

[0082] In an alternative embodiment, nucleic acids are detected in asample by DNA amplification using primer oligonucleotides selected fromSEQ ID NOS:2-18. One such amplification method is polymerase chainreaction (PCR) described in more detail below.

[0083] While high to moderately stringent hybridization conditions aregenerally used to detect and isolate variant nucleic acids of theinvention, nucleic acids that do not hybridize to each other understringent conditions are still substantially identical to the referencenucleic acids if the polypeptides they encode are substantiallyidentical. This may occur, e.g., when a copy of a nucleic acid iscreated using the maximum codon degeneracy permitted by the geneticcode. One indication that two nucleic acid sequences are substantiallyidentical is when the polypeptide encoded by the first nucleic acid isimmunologically cross reactive with the polypeptide encoded by thesecond nucleic acid. Hence, as is known to one of skill in the art, highto moderately stringent hybridization conditions can be used to detectand isolate substantially homologous to the reference nucleic acid, butsuch substantially homologous nucleic acids can also be detected orisolated with low stringency hybridization conditions.

[0084] Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific double-stranded sequence at a defined ionic strengthand pH. For example, under “highly stringent conditions” or “highlystringent hybridization conditions” a nucleic acid will hybridize to itscomplement to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). By controlling the stringency of thehybridization and/or washing conditions, nucleic acids that are 100%complementary can be identified.

[0085] Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Typically, stringent conditions will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide.

[0086] Exemplary low stringency conditions include hybridization with abuffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl and0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5×to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

[0087] The degree of complementarity or homology of hybrids obtainedduring hybridization is typically a function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. The type and length of hybridizing nucleicacids also affects whether hybridization will occur and whether anyhybrids formed will be stable under a given set of hybridization andwash conditions. For DNA-DNA hybrids, the T_(m) may be approximated fromthe equation of Meinkoth and Wahl Anal. Biochem. 138:267-284 (1984):

T _(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L

[0088] where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

[0089] An example of stringent hybridization conditions forhybridization of complementary nucleic acids that have more than 100complementary residues on a filter in a Southern or Northern blot is 50%formamide with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent conditions is 0.15M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see also,Sambrook, infra). Often, a high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example of mediumstringency for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at45° C. for 15 minutes. An example low stringency wash for a duplex of,e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes.For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.0MNa ion, typically about 0.01 to 1.0 M Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C.

[0090] Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

[0091] The following are examples of sets of hybridization/washconditions that may be used to detect and isolate homologous nucleicacids that are substantially identical to reference nucleic acids of thepresent invention: a reference nucleotide sequence desirably hybridizesto the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., desirably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate(SDS), 0.5 M NapO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 65° C.

[0092] In general, T_(m) is reduced by about 1° C. for each 1% ofmismatching. Thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired sequence identity. Forexample, if sequences with >90% identity are sought, the T_(m) can bedecreased 10° C. Generally, stringent conditions are selected to beabout 5° C. or more lower than the thermal melting point (T_(m)) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point(T_(m)); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal meltingpoint (T_(m)); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermalmelting point (T_(m)).

[0093] If the desired degree of mismatching results in a T_(m) of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it isdesirable to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, Part 1,Chapter 2 (Elsevier, N.Y. ); and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 2 (Greene Publishing andWiley—Interscience, New York). See also, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). Using these references and theteachings herein on the relationship between T_(m), mismatch, andhybridization and wash conditions, those of ordinary skill can generatevariants of the present dGTPase nucleic acids.

[0094] Computer analyses can also be utilized for comparison ofsequences to determine sequence identity. Such analyses include, but arenot limited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Version 8 (available from Genetics Computer Group(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using theseprograms can be performed using the default parameters. The CLUSTALprogram is described by Higgins et al. Gene 73:237 244 (1988); Higginset al. CABIOS 5:151-153 (1989); Corpet et al. Nucleic Acids Res.16:10881-90 (1988); Huang et al. CABIOS 8:155-65 (1992); and Pearson etal. Meth. Mol. Biol. 24:307-331(1994).

[0095] The BLAST programs are described by Altschul et al., J. Mol.Biol. 215:403 (1990). To obtain gapped alignments for comparisonpurposes, Gapped BLAST (in BLAST 2.0) can be utilized as described inAltschul et al. Nucleic Acids Res. 25:3389 (1997). Alternatively,PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.,supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the defaultparameters of the respective programs (e.g. BLASTN for nucleotidesequences, BLASTX for proteins) may be used. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA, 89, 10915 (1989)). See http://www.ncbi.nlm.nih.gov. Alignment mayalso be performed manually by inspection.

[0096] For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the dGTPasesequences disclosed herein is desirably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the desired program.

[0097] Enterobacteriaceae dGTPase Protein

[0098] The invention provides isolated dGTPase polypeptides from anyspecies of Enterobacteriaceae, as well as fragments thereof and variantdGTPase polypeptides that retain dGTPase activity. In one embodiment,the invention provides a polypeptide of SEQ ID NO:19, that is a wildtype Escherichia coli dGTPase polypeptide: 1 MAQIDFRKKI NWHRRYRSPQGVKTEHEILR IFESDRGRII NSPAIRRLQQ 51 KTQVFPLERN AAVRTRLTHS MEVQQVGRYIAKEILSRLKS LNTELTGPFE 101 SIVEYACLMH DIAIRRLVIL AKRTINDWFG QRLHPEDAESQPLTDRCSVA 151 ALRLRTGKNR LTSCGARFVR TYVILRGMHK HSPGAYIDAD ESHLGTGWRY201 FKIYPSGVVA CETPETHHYL MKKPGYYLSE EAYIARLRKE LNLALYSRFP 251LTWIMEAADD ISYCVADLED AVEKRIFTVE QLYHHLHEAW GQHEKGSLFS 301 LVVENAWEKSRSNSLSRSTE DQFFMYLRVN TLNKLVPYAA QRFIDNLPAI 351 FAGRFNHALL EDASECSDLLKLYKNVAVKH VFSHPDVERL ELQGYRVISG 401 LLEIYRPLLS LSLSDFTELV EKERVKRFPIESRLFHKLST PHRLAYVEAV 451 SKLPSDSPEF PLWEYYYRCR LLQDYISGMT DLYAWDEYRRLMAVEQ

[0099] In another embodiment, the invention provides a polypeptide ofSEQ ID NO:20, that is a wild type Salmonella typhimurium dGTPasepolypeptide: 1 MASIDFRNKI NWHRRYRSPQ GVKTEHEILR IFESDRGRLI NSPAIRRLQQ 51KTQVFPLERN AAVRTRLTHS MEVQQVGRYI AKEILSRLKE QDRLEEYGLD 101 ALTGPFESIVEMACLMHDIG NPPFGHFGEA AINDWFRQRL HPEDAESQPL 151 THDRCVVFSL RLQKYVRDICHLKACTREFV CTIRSCGGIL TWAAVRPNFK 201 NIPVPACWPR GRSRIPIRYL MKKPRYYLSEEKYIARLRKE LQLRPYSRFP 251 LTWIMEAADD ISYCVADLED AVEKRIFSVE QLYHHLYHAWCHHEKDSLFE 301 LVVGNAWEKS RANTLSRSTE DQFFMYLRVN TLNKLVPYAQ RFIDNLPQIF351 AGTFNQALLE DASGFSRLLE LYKNVAVEHV FSHPDVEQLE LQGYRVISGL 401LDIYQPLLSL SLNDFRELVE KERLKRFPIE SRLFQKLSTR HRLAYVEVVS 451 KLPTDSAEYPVLEYYYRCRL IQDYISGMTD LYAWDEYRRL MAVEQ

[0100] In another embodiment, the invention provides a polypeptide ofSEQ ID NO:21, that is a wild type Klebsiella oxytoca dGTPasepolypeptide: 1 MAKIDFRNKI NWRRRFRSPP RVETERDILR IFESDRGRIV NSPAIRRLQQ 51KTQVFPLERN GRVRTRLTHS LEVQQVGRYI AKEVLSRLKE LRLLEEYGLE 101 ELTGPFESVVEMACLMHDIG NPPFGHFGEA AINDWFRQRL APGDALGQPL 151 TDDRCEVQAL RLHDGETSLNALRRKVRQDL CSFEGNAQGI RLVHTLMRMN 201 LTWAQVGCIL KYTRPAWWSE ETPASHSYLMKKPGYYLAEE EYVARLRKEL 251 DLAPYNRFPL TWIMEAADDI SYCVADLEDA VEKRIFSAEQLYQHLYDAWG 301 SHVKRSRYSQ VVENAWEKSR ANYLKQSAED QF

[0101] The invention further provides peptides that are uniquely foundin certain Enterobacteriaceae bacterial species. These peptides arelisted below in Table 2. TABLE 2 Species Positions Sequence SEQ ID NO:Salmonella 141-147 HPDEAES 22 Escherichia 141-147 HPDEAES 22 Klebsiella141-147 APGDALG 23 Yersinia 141-147 DPNGGGA 24 Salmonella 156-159 VVFS25 Escherichia 156-159 SVAA 26 Klebsiella 156-159 EVQA 27 Yersinia156-159 LVNT 28 Salmonella 163-171 QEGEENLND 29 Escherichia 163-171RDGEEPLNE 30 Klebsiella 163-171 HDGETSLNA 31 Yersinia 163-171 REGETELNI32 Salmonella 221-227 RSRIPIR 33 Escherichia 221-227 ETPETHH 34Klebsiella 221-227 ETPASHS 35 Yersinia 221-227 DIPTSHN 36

[0102] The polypeptides and peptides of the invention are isolated orsubstantially purified polypeptides. In particular, the isolatedpolypeptides of the invention are substantially free of other proteinsnormally present in Enterobacteriaceae bacteria. Preparations andcompositions of the present polypeptides are substantially free ofcellular material. For example, such preparations and compositions haveless than about 30%, 20%, 10%, or 5%, (by dry weight) of contaminatingprotein.

[0103] By “variant” polypeptide is intended a polypeptide from anEnterobacteriaceae bacterial species other than E. coli or a polypeptidederived from any Enterobacteriaceae dGTPase by deletion or addition ofone or more amino acids to the N-terminal and/or C-terminal end of thedGTPase polypeptide; deletion or addition of one or more amino acids atone or more sites within the dGTPase polypeptide; or substitution of oneor more amino acids at one or more sites within the dGTPase polypeptide.Thus, the polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions.

[0104] Such variant polypeptides may result, for example, from geneticpolymorphism or from human manipulation. Methods for such manipulationsare generally known in the art. For example, amino acid sequencevariants of the polypeptides may be prepared by mutations in the DNA.Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA,82, 488 (1985); Kunkel et al., methods in Enzymol., 154, 367 (1987);U.S. Pat. No. 4,873,192; Walker and Gaastra, eds., Techniques inMolecular biology, MacMillan Publishing Company, New York (1983) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoffet al., Atlas of ProteinSequence and Structure, Natl. Biomed. Res. Found., Washington, C. D.(1978), herein incorporated by reference.

[0105] The variants of the isolated polypeptides of the invention haveidentity with at least about 60% of the amino acid positions of SEQ IDNO:19-36. In a desired embodiment, polypeptide variants have identitywith at least about 70% of the amino acid positions of SEQ ID NO:19-36.More desired polypeptide variants have at least about have identity withat least about 80% of the amino acid positions of SEQ ID NO:19-36. Evenmore desired polypeptide variants have at least about have identity withat least about 90% of the amino acid positions of SEQ ID NO:19-36. Mostdesired variant polypeptide variants have at least about have identitywith at least about 95% of the amino acid positions of SEQ ID NO:19-36.Especially desired variant polypeptide variants have at least about haveidentity with at least about 95% of the amino acid positions of SEQ IDNO:20-36. Such variants can have dGTPase activity and/or areimmunologically reactive with antibodies raised against the presentdGTPase polypeptides and peptides.

[0106] Amino acid residues of the isolated polypeptides and polypeptidevariants may be genetically encoded L-amino acids, naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids orD-enantiomers of any of the above. The amino acid notations used hereinfor the twenty genetically encoded L-amino acids and common non-encodedamino acids are conventional and are as shown in Table 3. TABLE 3One-Letter Amino Acid Symbol Common Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val β-Alanine bAla 2,3-Diaminopropionic acid Dprα-Aminoisobutyric acid Aib N-Methylglycine (sarcosine) MeGly OrnithineOrn Citrulline Cit t-Butylalanine t-BuA t-Butylglycine t-BuGN-methylisoleucine MeIle Phenylglycine Phg Cyclohexylalanine ChaNorleucine Nle Naphthylalanine Nal Pyridylalanine Abbreviation?3-Benzothienyl alanine Abbreviation? 4-Chlorophenylalanine Phe(4-Cl)2-Fluorophenylalanine Phe(2-F) 3-Fluorophenylalanine Phe(3-F)4-Fluorophenylalanine Phe(4-F) Penicillamine Pen 1,2,3,4-Tetrahydro- Ticisoquinoline-3-carboxylic acid β-2-thienylalanine Thi Methioninesulfoxide MSO Homoarginine hArg N-acetyl lysine AcLys 2,4-Diaminobutyric acid Dbu ρ-Aminophenylalanine Phe(pNH2) N-methylvaline MeValHomocysteine hCys Homoserine hSer ε-Amino hexanoic acid Aha δ-Aminovaleric acid Ava 2,3-Diaminobutyric acid Dab

[0107] Polypeptide variants that are encompassed within the scope of theinvention may have one or more amino acids substituted with an aminoacid of similar chemical and/or physical properties, so long as thesevariant polypeptides retain dGTPase activity and/or remainimmunologically reactive with antibodies raised against dGTPasepolypeptides having SEQ ID NO:19-36.

[0108] Amino acids that are substitutable for each other generallyreside within similar classes or subclasses. As known to one of skill inthe art, amino acids can be placed into three main classes: hydrophilicamino acids, hydrophobic amino acids and cysteine-like amino acids,depending primarily on the characteristics of the amino acid side chain.These main classes may be further divided into subclasses. Hydrophilicamino acids include amino acids having acidic, basic or polar sidechains and hydrophobic amino acids include amino acids having aromaticor apolar side chains. Apolar amino acids may be further subdivided toinclude, among others, aliphatic amino acids. The definitions of theclasses of amino acids as used herein are as follows:

[0109] “Hydrophobic Amino Acid” refers to an amino acid having a sidechain that is uncharged at physiological pH and that is repelled byaqueous solution. Examples of genetically encoded hydrophobic aminoacids include Ile, Leu and Val. Examples of non-genetically encodedhydrophobic amino acids include t-BuA.

[0110] “Aromatic Amino Acid” refers to a hydrophobic amino acid having aside chain containing at least one ring having a conjugated π-electronsystem (aromatic group). The aromatic group may be further substitutedwith substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl,sulfonyl, nitro and amino groups, as well as others. Examples ofgenetically encoded aromatic amino acids include phenylalanine, tyrosineand tryptophan. Commonly encountered non-genetically encoded aromaticamino acids include phenylglycine, 2-naphthylalanine,β-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and4-fluorophenylalanine.

[0111] “Apolar Amino Acid” refers to a hydrophobic amino acid having aside chain that is generally uncharged at physiological pH and that isnot polar. Examples of genetically encoded apolar amino acids includeglycine, proline and methionine. Examples of non-encoded apolar aminoacids include Cha.

[0112] “Aliphatic Amino Acid” refers to an apolar amino acid having asaturated or unsaturated straight chain, branched or cyclic hydrocarbonside chain. Examples of genetically encoded aliphatic amino acidsinclude Ala, Leu, Val and Ile. Examples of non-encoded aliphatic aminoacids include Nle.

[0113] “Hydrophilic Amino Acid” refers to an amino acid having a sidechain that is attracted by aqueous solution. Examples of geneticallyencoded hydrophilic amino acids include Ser and Lys. Examples ofnon-encoded hydrophilic amino acids include Cit and hCys.

[0114] “Acidic Amino Acid” refers to a hydrophilic amino acid having aside chain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Examples of genetically encoded acidic amino acids includeaspartic acid (aspartate) and glutamic acid (glutamate).

[0115] “Basic Amino Acid” refers to a hydrophilic amino acid having aside chain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Examples of genetically encoded basic amino acidsinclude arginine, lysine and histidine. Examples of non-geneticallyencoded basic amino acids include the non-cyclic amino acids ornithine,2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

[0116] “Polar Amino Acid” refers to a hydrophilic amino acid having aside chain that is uncharged at physiological pH, but that has a bond inwhich the pair of electrons shared in common by two atoms is held moreclosely by one of the atoms. Examples of genetically encoded polar aminoacids include asparagine and glutamine. Examples of non-geneticallyencoded polar amino acids include citrulline, N-acetyl lysine andmethionine sulfoxide.

[0117] “Cysteine-Like Amino Acid” refers to an amino acid having a sidechain capable of forming a covalent linkage with a side chain of anotheramino acid residue, such as a disulfide linkage. Typically,cysteine-like amino acids generally have a side chain containing atleast one thiol (SH) group. Examples of genetically encodedcysteine-like amino acids include cysteine. Examples of non-geneticallyencoded cysteine-like amino acids include homocysteine andpenicillamine.

[0118] As will be appreciated by those having skill in the art, theabove classification are not absolute. Several amino acids exhibit morethan one characteristic property, and can therefore be included in morethan one category. For example, tyrosine has both an aromatic ring and apolar hydroxyl group. Thus, tyrosine has dual properties and may beincluded in both the aromatic and polar categories. Similarly, inaddition to being able to form disulfide linkages, cysteine also hasapolar character. Thus, while not strictly classified as a hydrophobicor apolar amino acid, in many instances cysteine can be used to conferhydrophobicity to a peptide.

[0119] Certain commonly encountered amino acids that are not geneticallyencoded and that can be present, or substituted for an amino acid, inthe variant polypeptides of the invention include, but are not limitedto, β-alanine (b-Ala) and other omega-amino acids such as3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr),4-aminobutyric acid and so forth; α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine(MeGly); omithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); .beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid(Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH2));N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer).These amino acids also fall into the categories defined above.

[0120] The classifications of the above-described genetically encodedand non-encoded amino acids are summarized in Table 4, below. It is tobe understood that Table 4 is for illustrative purposes only and doesnot purport to be an exhaustive list of amino acid residues that maycomprise the variant polypeptides described herein. Other amino acidresidues that are useful for making the peptides and peptide analoguesdescribed herein may be found, e.g., in Fasman, 1989, CRC PracticalHandbook of Biochemistry and Molecular Biology, CRC Press, Inc., and thereferences cited therein. Amino acids not specifically mentioned hereincan be conveniently classified into the above-described categories onthe basis of known behavior and/or their characteristic chemical and/orphysical properties as compared with amino acids specificallyidentified. TABLE 4 Genetically Classification Encoded GeneticallyNon-Encoded Hydrophobic F, L, I, V Aromatic F, Y, W Phg, Nal, Thi, Tic,Phe(4-Cl), Phe(2- F), Phe(3-F), Phe(4-F), Pyridyl Ala, Benzothienyl AlaApolar M, G, P Aliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal,Cha, bAla, MeGly, Aib Hydrophilic S, K Cit, hCys Acidic D, E Basic H, K,R Dpr, Orn, hArg, Phe(p-NH2), DBU, A2 BU Polar Q, N, S, T, Y Cit, AcLys,MSO, hSer Cysteine-Like C Pen, hCys, β-methyl Cys

[0121] Polypeptides of the invention can have any amino acid substitutedby any similarly classified amino acid to create a variant peptide, solong as the peptide variant retains dGTPase activity and/or isimmunologically reactive with antibodies raised against dGTPasepolypeptides having any one of SEQ ID NO:19-36.

[0122] Thus, the polypeptides of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired activity. Thedeletions, insertions, and substitutions of the polypeptide sequenceencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. One skilled in the art can readilyevaluate the thermal stability and dGTPase activity of the polypeptidesand variant polypeptides of the invention by routine screening assays.

[0123] The activity of dGTPases and variant dGTPase polypeptides can beassessed by any procedure known to one of skill in the art. For example,the method described by Seto et al. (13) can be used, which involvesmeasuring the hydrolysis of dGTP to PPPi. In general, a mixture isprepared with dGTP, MgCl2 and the dGTPase polypeptide to be tested.After incubation at 37° C., the reaction is terminated, the mixture iscentrifuged, and an aliquot of the supernatant acidified, and boiled.The acidification and boiling hydrolyzes the tripolyphosphate toorthophosphate. The total orthophosphate concentration can then bedetermined by any available method. For example, orthophosphate can bedetected colorimetrically by the addition of an ascorbate-molybdatesolution, which gives rise to a product detectable at 780 nm.

[0124] Preparation of dGTPase Polypeptides

[0125] Methods are readily available to those skilled in the art forconstructing expression cassettes and vectors containing a nucleic acidof the invention that encodes a dGTPase polypeptide with appropriatetranscriptional/translational control signals. For example, in vitrorecombinant DNA techniques, synthetic techniques and in vivorecombination/genetic techniques can be used to make expressioncassettes and vectors. See, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (2d ed.) (1989) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (3ded.) (2001).

[0126] Generally, an expression cassette is in the form of chimeric DNAthat can be part of an expression vector comprising a plasmid DNA andthe nucleic acids encoding the polypeptide(s) of interest, flanked bycontrol sequences that promote the expression, or stop the expression,of the DNA segment encoding the polypeptide. Aside from nucleic acidsthat serve as expression cassettes for dGTPase polypeptides, a portionof the expression vector may be untranscribed, serving a regulatory,replication or a structural function. Additional transcribed portions ofthe expression vector can include selectable markers, vector-specificreplication functions, and the like. Other elements functional in thehost cells, such as enhancers, polyadenylation sequences and the like,may also be a part of the expression vector. Such elements can provideimproved expression of the dGTPase nucleic acids by affectingtranscription, mRNA stability, or another factor influencing theperformance of the expression vector in a host cell.

[0127] Thus, depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, and the like can be used in the expressionvector (see, e.g., Bitter et al., 1987; WO 97/11761 and WO 96/06167).For example, when cloning in bacterial systems, inducible promoters suchas pL of bacteriophage λ; plac, ptrup, ptac (ptrp-lac hybrid promoter)and the like may be used. Promoters produced by recombinant DNA orsynthetic techniques can also be used to provide for controlled and highlevel transcription of the inserted coding sequence.

[0128] The expression cassette may encode other peptides. For example,an expression cassette comprising an isolated nucleic acid of theinvention can be fused in-frame to another nucleic acid encoding apeptide, polypeptide or protein, which is expressed as a fusion proteincontaining at least a segment of a dGTPase polypeptide. In thisembodiment, the isolated nucleic acid includes a first DNA segmentencoding an immunogenic dGTPase peptide and a second DNA segmentencoding a carrier protein. The carrier protein can facilitatepurification of the resulting fusion polypeptide and/or can helpactivate T helper cells. The carrier protein desirably possesses lowimmunoreactivity.

[0129] The expression cassette to be introduced into the cells can alsocontain either a selectable marker gene or a reporter gene, or both, tofacilitate identification and selection of transformed cells from thepopulation of cells sought to be transformed. Alternatively, theselectable marker may be carried on a separate piece of DNA and used ina co-transformation procedure. Both selectable markers and reportergenes may be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers are well knownin the art and include, for example, antibiotic-resistance genes.

[0130] Reporter genes are used for identifying transformed cells and forevaluating the functionality of regulatory sequences. Desirable reportergenes encode polypeptides that can be easily assayed. Many such reportergenes are known in the art. In general, a reporter gene is a gene thatis not present in or expressed by the recipient organism or tissue andthat encodes a polypeptide whose expression is manifested by some easilydetectable property, e.g., enzymatic activity. Desired genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, thebeta-glucuronidase gene (gus) of the uidA locus of E. coli, and theluciferase gene (luc) from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

[0131] Expression vectors can be readily introduced into host cells,e.g., mammalian, plant, bacterial, yeast or insect cells by transfectionwith an expression cassette by any procedure useful for the introductioninto a particular cell, e.g., calcium phosphate precipitation,lipofection, microinjection, electroporation, and the like, to yield atransformed cell, so that the peptide, e.g., fusion protein, of thepresent invention is expressed by the host cell.

[0132] General methods for isolating and purifying a recombinantlyexpressed protein from a host cell are available to those in the art.For example, the culture medium or lysate can be centrifuged to removeparticulate cell debris. The insoluble and soluble polypeptide fractionsare then separated. The peptide of the invention may then be purifiedfrom the soluble fraction or the insoluble fraction, i.e., refractilebodies (see, for example, U.S. Pat. No. 4,518,526, the disclosure ofwhich is incorporated by reference herein). The peptide can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, Scopes, 1982; Deutscher1990). Substantially pure compositions of at least about 90 to 95%homogeneity are desirable, and compositions of 98 to 99% or morehomogeneity are more desirable. Examples of the isolation andpurification of recombinant polypeptides and proteins are given inSambrook et al., cited supra.

[0133] For example, the dGTPase polypeptides of the invention can beprepared from enteric or recombinant bacterial cells collected bycentrifugation. After washing in buffer, the cells can be disrupted bysonication and the sonicate clarified by centrifugation. Because thedGTPases of the invention are thermostable, the supernatant from thesonication step can be heated at about 60° C. and precipitated proteinsremoved by centrifugation. The supernatant containing dGTPasepolypeptides can be further purified using a DEAE Sepharose column withelution using a gradient of KCl. Further purification can be achievedusing a single-stranded DNA cellulose column. After applying thesemi-purified dGTPase preparation and washing off impurities with a KClsolution, active dGTPase can be eluted from the column with using higherconcentration of KCl. The dGTPase enzyme can also be concentrated viapressure filtration and dialyzed into new buffers needed.

[0134] The activity of dGTPases can be assessed by any procedure knownto one of skill in the art. For example, the method described by Seto etal. (13) can be used that involves measuring the hydrolysis of dGTP toPPPi. In general, a mixture is prepared with dGTP, MgCl₂ and the dGTPaseto be tested. After incubation at 37° C., the reaction was terminated,the mixture was centrifuged, and an aliquot of the supernatantacidified, and boiled. The acidification and boiling hydrolyzes thetripolyphosphate to orthophosphate. The total orthophosphateconcentration can then be determined by any available method. Forexample, orthophosphate can be detected colorimetrically by the additionof an ascorbate-molybdate solution, which gives rise to a productdetectable at 780 nm.

[0135] Antibodies

[0136] The present invention also provides antibodies directed againstany Enterobacteriaceae dGTPase including, for example, any one of SEQ IDNO:19-36. More desirable antibody preparations are directed against apeptide or polypeptide having any one of any one of SEQ ID NO:20-36.Immunologically active fragments of the present antibodies are alsowithin the scope of the present invention, e.g., F(ab) fragments.

[0137] Antibodies may be prepared by any of a variety of techniquesknown to those of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Inone such technique, an immunogen comprising an entire dGTPasepolypeptide or an antigenic portion of a dGTPase polypeptide is injectedinto any of a wide variety of mammals (e.g., birds, mice, rats, rabbits,sheep and goats). The immunogen can be bound to a carrier peptide and/oremulsified using a biologically suitable emulsifying agent, such asFreund's incomplete adjuvant. The immunogen is injected into the animalhost, desirably according to a predetermined schedule incorporating oneor more booster immunizations.

[0138] After immunization, the animals are bled periodically, bloodcells are removed and the serum (antiserum) can be used as a source ofpolyclonal antibodies. Polyclonal antibodies can be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

[0139] Monoclonal antibodies specific for the antigenic polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells may then be immortalizedby fusion with a myeloma cell fusion partner, desirably one that issyngeneic with the immunized animal, using one of a variety oftechniques well known in the art.

[0140] Monoclonal antibodies may be isolated from the supernatants ofthe resulting hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood of that host.

[0141] Monoclonal antibodies offer certain advantages in comparison topolyclonal antibodies. In particular, monoclonal antibodies are highlyspecific and sensitive and relatively “pure” immunochemically. Amonoclonal antibody also may be subjected to the techniques ofrecombinant DNA technology to produce other derivative antibodies,humanized or chimeric molecules or antibody fragments that retain thespecificity of the original monoclonal antibody. Such techniques mayinvolve combining DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of the monoclonalantibody with DNA coding the constant regions, or constant regions plusframework regions, of a different immunoglobulin, for example, toconvert a mouse-derived monoclonal antibody into one having largelyhuman immunoglobulin characteristics (see, for example, EP 184187A andEP 2188638A, herein incorporated by reference).

[0142] Biosensors

[0143] The antibodies or nucleic acids of the invention can be absorbedor attached to a solid support or substrate to facilitate detection ofEnterobacteriaceae. Such articles are useful for identifying which genusor species of bacteria is present within a test sample. Solid supportsor substrates may be biosensors or a dipsticks. Antibodies and nucleicacids are bound to the support in an amount and manner that allowsbinding of dGTPase polypeptides or complementary nucleic acids. Theamount of the antibodies and nucleic acids used relative to a givensubstrate depends upon the particular antibody or nucleic acid beingused, the particular substrate, and the antibody-polypeptide binding ornucleic acid hybridization efficiency.

[0144] The antibodies and nucleic acids of the invention may be bound tothe substrate in any suitable manner. Covalent, noncovalent, or ionicbinding may be used. Covalent bonding can be accomplished by attachingthe antibodies or nucleic acids to reactive groups on the substratedirectly or through a linking moiety.

[0145] The solid support may be any insoluble material to which theantibodies or nucleic acids can be bound and that may be convenientlyused in the assay of the invention. The biosensor or solid support canbe formed from a rigid support. Alternatively, the antibodies or nucleicacids may be bound to any porous or liquid permeable material, such as afibrous (paper, felt, nitrocellulose and the like) strip or sheet, or ascreen or net, or any matrix material that one of skill in the art canconveniently use in an assay for Enterobacteriaceae. Such solid supportsinclude permeable and semipermeable membranes, quartz, glass beads,plastic beads, latex beads, plastic microtiter wells or tubes, agaroseor dextran particles, sepharose, and diatomaceous earth. The substrateof the solid support may be functionalized glass, quartz, Si, Ge, GaAs,GaP, SiO₂, SiN₄, modified silicon, or any one of a wide variety of gelsor polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other substrate materials will be readily apparent to thoseskilled in the art upon review of this disclosure. In a desirableembodiment the substrate is quartz, flat glass, silica or silicon wafer.

[0146] The surface of the solid substrate or chip can be coated withother materials, for example, polymers, plastics, resins,polysaccharides, carboxymethyldextran, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or any of the above-listedsubstrate materials. In one embodiment, the substrate or chip is asilicon wafer coated with one or more metals and then coated with apolysaccharide. For example, the substrate or chip can be coated withgold, silver, chromium and/or combinations or layers of metal, such aschromium and gold. Metal depositions can be carried out byelectro-deposition, by use of a sputtering device or by use of acryo-pumped evaporator at reduced air pressure. The metal coat can thenbe coated with a polysaccharide such as carboxymethyldextran.

[0147] Distinct types of antibodies and nucleic acids can be arrayed ona substrate or chip in addressable rows and columns. Procedures anddevices are available to read information from such arrays of rows andcolumns. For easy and/or automated detection, the size of the separateantibody or nucleic acid loci is uniform. For example, each locus can bea square, rectangle or other simple geometric shape with an area ofabout one square micron to about 500 square microns. The size of theloci can vary depending on the specificity and affinity of the adsorbedor immobilized antibody, the density of the antibody at a given locusand the strength of the signal or the sensitivity of the detectionprocedure. Similarly, the size of the loci can vary with the degree ofcomplementarity between the nucleic acid probe and target, the densityof the probe molecules on the substrate and the sensitivity of thedetection procedure.

[0148] Antibodies and nucleic acid probes may be absorbed, attached orimmobilized on the substrate or chip by any available procedure known toone of skill in the art. The selected antibody or probe may beimmobilized to the substrate, support or chip by adsorption or covalentattachment. If covalent coupling is chosen, a wide variety of knownmethods can be employed including derivatizing the support with thefollowing functional groups: benzoyl azide, bromoacetamide, azidoaryl,aldehyde, isothiocyanate, diazonium salt, acid chloride, active ester,iminocarbonate, hydrazide, epoxy, and amine.

[0149] In a desirable embodiment, a biosensor with attached nucleic acidor anti-dGTPase antibodies is used with detection by surface plasmonresonance (SPR). The selected antibodies or nucleic acids are coupled tothe surface of a gold coated quartz substrate. Test samples which maycontain dGTPase polypeptides or nucleic acids are applied and allowed toflow over the surface of the biosensor. After rinsing the surface of thebiosensor, bound dGTPase polypeptides or nucleic acids are detected bymeasuring the developing surface plasmon.

[0150] Although the methods of the invention can be used with anydetection strategy, including, for example, lateral flow assays, PCR,ELISA, and enzymatic detection, the surface plasmon resonance techniqueoffers higher detection sensitivity and speed than any other detectionstrategy (32-34). The biosensor-surface plasmon resonance technique candetect nanogram quantities of the dGTPase enzyme. Such detectionsensitivity is an order of magnitude better than published PCR baseddetection methods and results can be obtained in about half the time(29-31).

[0151] In one embodiment, a CM5 unmodified biosensor chip from BiaCore,Inc. is used. This chip is an approximate 1 cm square quartz crystalthat was coated with a thin gold layer. Attached to the gold layer was atether of carboxymethyldextran. The terminal carboxyl groups of thecarboxymethyldextran are easily modified in order to create reactivegroups for protein or nucleic acid coupling. For example, the chipsurface can be reacted with a solution of 100 mMN-ethyl-N′(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 50mM N-hydroxysuccinimide (NHS) in 25 mM sodium bicarbonate (pH 8.5) for 5minutes, followed by a brief rinse with 100 mM borate (pH 8.5). Thisprovides an activated CM5 chip. The activated chip can then be incubatedin 80 mM 2-(2-pyridinyldithio)ethaneamine (PDEA), 0.1 M borate (pH 8.5)for four minutes, followed by a brief rinse in 0.1 M borate (pH 8.5).The chip is then placed in contact with a solution of purifiedantibodies or nucleic acids. Finally, all reactive disulfides aredeactivated, and non-covalently bound protein or nucleic acids areremoved by soaking the chip surface in a cysteine solution containingsalt (e.g. NaCl).

[0152] Hence, the present invention provides a biosensor that includes asolid substrate with an antibody or a nucleic acid adsorbed orcovalently attached thereto. The solid substrate of the biosensor canhave a pattern or array of different antibody or nucleic acid probetypes. The surface of the biosensor can also have different antibody ornucleic acid probe concentrations adsorbed or covalently attachedthereto. Such an array can have a density of at least about 10 antibodyor nucleic acid probe loci per square cm, and up to about one millionantibody or nucleic acid probe loci per square centimeter.

[0153] Any surface plasmon resonance device can be used for detection ofbound antigen or nucleic acids. In one embodiment, a Texas Instrumentsportable SPR instrument (TISPR-1) is desirable because the entiredetection procedure can be performed in the field, by relativelyuntrained personnel, using a variety of sensor chips that containdifferent anti-dGTPase antibodies or nucleic acids depending on theneeds of the inspector or the consumer.

[0154] dGTPase Detection Methods

[0155] The dGTPase nucleic acids and/or the dGTPase enzymes of theinvention can serve as a key basis for a method of detectingEnterobacteriaceae and for a diagnostic device to facilitate performingthat method. In general, any procedure known to one of skill in the artfor detecting a nucleic acid or protein can be used for detecting thedGTPase polypeptides and nucleic acids described herein. For example,any molecular biology technique can be used, including immunoassay,hybridization or PCR procedures. Biophysical detection procedures can beused coupled with such procedures, or used separately as dictated by oneof skill in the, for example, procedures such as surface plasmonresonance, fluorescence, lateral flow procedures. These proceduresproduce a robust and useful means of detecting and identifying entericbacterial contamination in test samples.

[0156] In one embodiment, the invention provides a method for detectingEnterobacteriaceae in a test sample that involves contacting an antibodycapable of binding to a dGTPase polypeptide isolated from anEnterobacteriaceae bacteria with a test sample and detecting whether adGTPase polypeptide from the test sample has bound to the antibody. Sucha detection method is conducted at a temperature and under conditionssufficient for antigen-antibody interaction.

[0157] In another embodiment, the invention provides a method fordetecting Enterobacteriaceae in a test sample that involves contacting anucleic acid probe with the test sample under stringent hybridizationconditions and detecting whether the nucleic acid probe has hybridizedwith a dGTPase nucleic acid in the test sample. Nucleic acids useful asprobes for Enterobacteriaceae include any of the nucleic acids providedby the invention, for example, nucleic acids having SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0158] In one embodiment, the invention provides probes that can detectthe presence of any species of enteric bacteria (Enterobacteriaceae) ina test sample, for example, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6. However, the invention also provides probes andmethods for detecting one or more selected genus of bacteria within thefamily of Enterobacteriaceae. Such probes and methods are useful foridentifying which species of Enterobacteriaceae is present in a testsample. Thus, for example, probes made from nucleic acids having SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 can selectivelyhybridize to DNA from Escherichia coli in the presence of DNA fromKlebsiella, Salmonella, Shigella or Yersinia. Probes made from nucleicacids having SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14can selectively hybridize to DNA from Salmonella typhymurium, in thepresence of DNA from Klebsiella or Escherichia. Probes made from nucleicacids having SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18can selectively hybridize to DNA from Klebsiella oxytoca in the presenceof DNA from Salmonella or Escherichia.

[0159] The detection methods of the invention are not limited tohybridization and PCR methods but also include any immunological orimmunoassay method that one of skill in the art can adapt for use withthe present antibodies and dGTPase polypeptides. For example, theinvention includes a method for detecting enteric bacteria in a testsample that includes, contacting a test sample with a biosensor chipthat comprises a solid support and an antibody that can bind to dGTPasefrom Enterobacteriaceae; and detecting whether dGTPase is bound to thebiosensor chip.

[0160] Test samples which can be used in the present hybridization,immunological and PCR procedures include, for example, physiologicalfluids and samples from humans or animals, food samples, water, soil, aswell as samples taken from work areas, counter-tops, shelving, storageareas for food, animal or poultry pens, or from the skin, hair, orsurface of an animal. Such applications include human disease statetesting.

[0161] Antibodies may be used in diagnostic tests to detect the presenceof Enterobacteriaceae and species or genuses thereof, by binding todGTPase antigens. Any antibody-antigen procedure known by to one ofskill in the art can be adapted for use with the present antibodies anddGTPase polypeptides. Immunoassays contemplated by the inventiondesirably involve use of the present biosensors, but other antibodypreparations and procedures can also be used, such as those involvingradioimmunoassay, ELISA, or an immunofluorescence assay. Thus, forexample, immunoassays that are suitable for detecting an antigen such asthe dGTPase polypeptides of the invention include those described inU.S. Pat. Nos. 3,791,932; 3,817837; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; and 4,098,876.

[0162] In one embodiment, the invention provides a method for detectingEnterobacteriaceae by contacting antibodies of the invention with thesample for a period of time and under conditions sufficient forantibodies to bind to the dGTPase polypeptide so as to form a binarycomplex between at least a portion of the antibody and a portion of thedGTPase polypeptide. Such times, conditions and reaction media can bereadily determined by persons skilled in the art. For example, to testfor the presence of Enterobacteriaceae bacterial cells, a portion of thesample may be cultured and another portion may be lysed to yield anextract that comprises cellular proteins. The anti-dGTPase antibodiesare then incubated with the protein extract, e.g., using the biosensorchips provided herein, or a Western blot, or any other preparation ofantibodies provided herein that is capable of forming a detectablecomplex with an Enterobacteriaceae dGTPase. The presence or amount ofthe complex is then determined or detected, e.g., through determinationor detection of a label.

[0163] Optionally, a blocking agent is employed prior to the addition ofthe antibodies, or when the antibodies are added to the test sample, orafter rinsing away any un-bound antibodies, to block any non-specificbinding sites with a protein such as bovine serum albumin (BSA), ifdesired. A second reagent, e.g., an antibody that binds to the Fc of theprimary antibody, can then be added. After this second incubation, anyunreacted antibody is removed as by rinsing. Hence, a ternary complex ofantigen, primary antibody and secondary antibody is formed. The secondantibody may be labeled to facilitate detection of the ternary complex.Such a label can be, for example, a fluorescent dye. Alternatively, theprimary or secondary antibody may bind a detectable label.

[0164] Detection or measuring the formation of such complexes can alsoinclude a reagent capable of binding to the complexes formed by thedGTPase polypeptides and antibodies, and/or a label, reporter moleculeor other detectable moiety. Such a reagent may be an antibody of theinvention or an antibody that can bind to an antibody of the invention,that is conjugated to a detectable label, or reporter molecule.

[0165] The use of whole, intact antibodies is not necessary for manyimmunoassays. Instead, the antigen binding site alone may be used orsingle chain recombinant antibodies can be used. Examples of suchantibody combining sites are those known in the art as Fab and F(ab′)₂antibody portions that are prepared by proteolysis using papain andpepsin, respectively, as is well known in the art.

[0166] In one embodiment, the dGTPase nucleic acids of the invention areused in procedures involving DNA amplification. Any such DNAamplification procedure can be used, for example, in polymerase chainreaction (PCR) assays, strand displacement amplification and otheramplification procedures. Strand displacement amplification can be usedas described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696. Theterm “polymerase chain reaction” (“PCR”) refers to the method of K. B.Mullis U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, herebyincorporated by reference. These references by Mullis describe a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic or other DNA without cloning or purification.

[0167] The PCR process for amplifying a target sequence consists ofintroducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To do amplification, the mixture is denaturedand the primers are annealed to complementary sequences within thetarget molecule. Following annealing, the primers are extended with apolymerase so as to form a new pair of complementary strands. The stepsof denaturation, primer annealing and polymerase extension are termed a“cycle.” There can be numerous cycles, and the amount of amplified DNAproduced increases with each cycle. Hence, to obtain a highconcentration of an amplified target nucleic acid, many cycles areperformed.

[0168] The steps involve in PCR nucleic acid amplification method aredescribed in more detail below. For ease of discussion, the nucleic acidto be amplified is described as being double-stranded. However, theprocess is equally useful for amplifying a single-stranded nucleic acid,such as an mRNA, although the ultimate product is generallydouble-stranded DNA. In the amplification of a single-stranded nucleicacid, the first step involves the synthesis of a complementary strandusing, for example, one of the two amplification primers. The succeedingsteps generally proceed as follows:

[0169] (a) Each nucleic acid strand is contacted with four differentdeoxynucleoside triphosphates and one oligonucleotide primer for eachnucleic acid strand to be amplified, wherein each primer is selected tobe substantially complementary to a portion the nucleic acid strand tobe amplified, such that the extension product synthesized from oneprimer, when it is separated from its complement, can serve as atemplate for synthesis of the extension product of the other primer. Topromote the proper annealing of primer(s) and the nucleic acid strandsto be amplified, a temperature that allows hybridization of each primerto a complementary nucleic acid strand is used.

[0170] (b) After primer annealing, a DNA polymerase is used for primerextension that incorporates the nucleoside triphosphates into a growingnucleic acid strand that is complementary to the strand hybridized bythe primer. In general, this primer extension reaction is performed at atemperature and for a time effective to promote the activity of theenzyme and to synthesize a “full length” complementary nucleic acidstrand, that extends into a through a complete second primer binding.However, the temperature is not so high as to separate each extensionproduct from its nucleic acid template strand.

[0171] (c) The mixture from step (b) is then heated for a time and at atemperature sufficient to separate the primer extension products fromtheir complementary templates. The temperature chosen is not so high asto irreversibly denature the DNA polymerase present in the mixture.

[0172] (d) The mixture from (c) is cooled for a time and at atemperature effective to promote hybridization of a primer to each ofthe single-stranded molecules produced in step (b).

[0173] (e) The mixture from step (d) is maintained at a temperature andfor a time sufficient to promote primer extension by DNA polymerase toproduce a “full length” extension product. The temperature used is notso high as to separate each extension product from the complementarystrand template. Steps (c)-(e) are repeated until the desired level ofamplification is obtained.

[0174] The invention also comprises reagents and kits for detecting thepresence of dGTPase nucleic acids or polypeptides in a sample. Onereagent or kit can comprise purified antibodies of the invention in aliquid that does not adversely affect the activity of the antibodies inthe intended assay, for example, a saline solution. Another reagent orkit can comprise isolated nucleic acids of the invention in a liquidthat does not adversely affect the hybridization of the nucleic acids inthe intended assay. Yet another reagent or kit can comprise isolatedpolypeptides of the invention in a liquid that does not adversely affectthe activity of those polypeptides in the intended assay. Alternatively,the reagent or kit may comprise the purified antibodies, polypeptides ornucleic acids attached to a substrate as discussed above. Desiredsubstrates are insoluble solid supports, e.g., the biosensors describedherein or the well(s) of a microtiter plate.

[0175] Thus, in one embodiment the present invention provides a kit thatincludes a biosensor or solid support of the invention with an attachedantibody that is capable of binding to a dGTPase of anEnterobacteriaceae. The kit can contain control samples. Control samplesinclude, for example, one or more samples of EnterobacteriaceaedGTPases. The kit can also contain solutions for conducting the methodsof the invention, for example, solutions for diluting test samples, forincubating test samples with the biosensor or antibody-solid support,and for washing off any unbound test sample. The kit may also comprise ablocking agent that is contacted with the biosensor prior to or duringcontact with the sample. Desired control and other solutions are sterileand free of substances that may interfere with detection of bounddGTPase.

[0176] In another embodiment, the present invention provides a kit thatincludes a biosensor or solid support of the invention with an attachednucleic acid probe that is capable of binding to a dGTPase nucleic acidin a test sample which may contain one or more species ofEnterobacteriaceae. The kit can contain control samples. Control samplesinclude, for example, one or more samples of Enterobacteriaceae dGTPasenucleic acids. The kit can also contain solutions for conducting themethods of the invention, for example, solutions for diluting testsamples, for incubating test samples with the biosensor or solidsupport, and for washing off any unbound test sample. Desired controland other solutions are sterile and free of substances that mayinterfere with detection of dGTPase nucleic acids.

[0177] A label or reporter molecule that permits the antigen-antibody orthe hybridization complex can also be provided with any of the kits ofthe invention. Such a label or reporter molecule can be packagedseparately from the biosensor, nucleic acid or antibody.

[0178] An illustrative diagnostic system in kit form embodying oneaspect the present invention that is useful for detecting dGTPasenucleic acids, is at least one container or vial containing a nucleicacid probe of the invention, for example, a nucleic acid having one ofmore of SEQ ID NO:2-18.

[0179] Labeled Nucleic Acids, Polypeptides and Antibodies

[0180] The invention utilizes labeled antibodies, labeled nucleic acidprobes, and labeled dGTPase polypeptides. Labels that may be employedinclude radionuclides, fluorescent labels, chemiluminescent labels,colorimetric dyes, enzymes, enzyme substrates, enzyme cofactors, enzymeinhibitors, enzyme subunits, metal ions, particles, and the like.Radioisotopes commonly used as reporter molecules or labels include ³²P,¹²⁵I and ¹³¹I. Enzymes commonly used as reporter molecules or labelsinclude such as alkaline phosphatase, horseradish peroxidase,beta-D-galactosidase and glucose oxidase. Commonly used fluorescentreporter molecules or labels include, for example, dyes such asfluorescein isothiocyanate (FITC), fluorescein, rhodamine, rhodamine Bisothiocyanate (RITC), tetramethylrhodamine isothiocyanate (TRITC),4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). See, forexample, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.Other commonly used types of labels or reporter molecules include Texasred, phycoerythrin, umbelliferone, luminol, NADPH, and the like.

[0181] Various techniques can be employed for detecting and quantifyingthe presence of the label which are dependent upon the nature of thelabel. For fluorescent labels, a large number of different fluorometersand flourescent microscopes are available. For chemiluminescent labels,luminometers or films are available. Enzymes producing a fluorescent,chemiluminescent, or colored product can be detected fluorometrically,luminometrically, spectrophotometrically or visually. Such labels can beemployed in immunoassays and hybridization assays described herein.

[0182] Various means for providing labels bound to nucleic acids havebeen reported, e.g., Leary et al., Proc. Natl. Acad. Sci. (USA) (1983)80:4045; Renz and Kurz, Nucl. Acid Res. (1984) 12:3435; Richardson andGumport, Nucl. Acid Res. (1983) 11:6167; Smith et al., Nucl. Acid Res.(1985) 13:2399; and Meinkoth and Wahl, Anal, Biochem. (1984) 138:267.The labels may be bound either covalently or non-covalently to thecomplementary sequence. Thus, for example, various labels may beattached to an antibody, nucleic acid or polypeptide via a carboxy,thiol, amine, hydrazine or other functionality without detrimentallyaffecting complex formation or hybridization of those entities.

[0183] The invention will be further described by the followingexamples.

EXAMPLE 1 Materials and Methods

[0184] General Procedures

[0185] All bacterial strains were purchased from the American TypeCulture Collection (ATCC). The ATCC deposit numbers of the strains arelisted in Table 5. Bacterial growth conditions and culturing wereperformed as described by Miller (18). Protein concentration wasdetermined according to the method of Bradford (19) using bovine serumalbumin (BSA) as a standard or spectrophotometrically using a calculatedmolar absorption coefficient of 86,300 M⁻¹ cm⁻¹ (20). All proteinconcentrations were for the dGTPase tetramer.

[0186] Analytical gel filtration experiments were performed according toSiegel and Monty (21) using Sephacryl S-300. Protein SDS PAGE gels weremade, run, and processed as per Laemmli (22).

[0187] Single-stranded DNA binding assays were performed according toWurgler and Richardson (23). Polymerase chain reaction (PCR)amplification of DNA was performed using VENT thermopolymerase from NewEngland Biolabs, Inc. according to the instructions provided by themanufacturer. All PCR reactions were carried out in a ProGenethermocycler from Techne, Inc.

[0188] Enzyme Assay Procedures

[0189] Enzyme assay procedures were performed in a manner similar tothose described by Seto et al. (13) by measuring the hydrolysis of dGTPto Norit non-adsorbable PPPi. The incubation mixture had a volume of 55μL and included 2.0 mM dGTP; 67 mM Glycine, pH 8.5; 6.7 mM MgCl2; and0.02-0.2 milliunit of enzyme. After 20 min at 37° C., the reaction wasterminated by addition of an acidic suspension of Norit A. The mixturewas centrifuged, and an aliquot of the supernatant was brought to 0.15 NHCl, and was boiled for 15 minutes. This step in the procedure resultedin the hydrolysis of tripolyphosphate to orthophosphate. The totalorthophosphate concentration was determined calorimetrically by theaddition of an ascorbate-molybdate solution. The absorbance at 780 nmwas determined after incubating the mixture at 45° C. for 15 minutes.Results were compared to a phosphate standard curve. A unit of enzymeforms 1 μmol of tripolyphosphate under these conditions. The stimulatoryeffect of ssDNA was assayed by adding 50 μg/mL of m13 mp18 DNA to thestandard assay.

[0190] Purification of Native E. coli dGTPase

[0191] Cells were grown at 37° C. in Luria broth from a 1% innoculum forten hours before harvesting. Typically, 4 g of cells were obtained perliter. Cells were pelleted by centrifugation at 10,000×g for ten minutesand resuspended in one volume of 10 mM Tris-HCl, pH 8.0. The cells wererespun as above and resuspended in two volumes of 10 mM Tris, pH 8.0, 1mM EDTA, 1 mM sodium azide (Buffer I). The cells were disrupted bysonication on ice using a Branson sonicator. The extract was clarifiedby centrifugation at 12,000×g for 20 minutes, and the supernatant wasdesignated as Fraction I. All subsequent chromatography steps wereperformed at room temperature.

[0192] Fraction I was heated in a water bath at 60° C. for 20 minuteswith gentle swirling. During this period a large precipitate formed.After this heat treatment, the protein solution was immediately cooledto 4° C. in an ice bath. The supernatant was clarified by two successivecentrifugations at 15,000×g for 30 minutes each. The protein in thedecanted supernatant fraction from the second centrifugation step wasdesignated as Fraction II.

[0193] Fraction II was applied to a 4.9 cm²×30 cm column of DEAESepharose (Sigma Chemical Co.) in Buffer I. The enzyme was eluted fromthe column with a concave gradient of 0 to 0.5 M KCl at a flow rate of2.0 ml/min. The major peak containing dGTPase activity eluted as a broadpeak centered at 0.35 M KCl. The central 90% of the activity peak waspooled and constituted Fraction III.

[0194] This material was immediately applied to a single-stranded DNAcellulose column (4.9 cm²×15 cm) that was equilibrated in Buffer I.After loading, the column was washed with five column volumes of BufferI followed by 10 column volumes of Buffer I, 1.0 M KCl. Active dGTPasewas eluted from the column with 10 column volumes of Buffer I, 3.0 MKCl. The homogeneous enzyme was concentrated via pressure filtrationthrough a semipermeable membrane (Amicon YM 3) and dialyzed into newbuffer constituents as needed. Purification of native S. marcescensdGTPase Enzyme from Serratia cultures was prepared to Fraction III asdescribed above. Because this enzyme cannot bind to ssDNA, analternative purification strategy was employed. Fraction III was loadedonto a prepacked Mono Q column (BioRad Labs Inc., column volume 1.0 mL),which was equilibrated with Buffer I. Elution was with a 50 mL linearzero to 1.0 M NaCl gradient at a flow rate of 3.0 mL/min. FunctionaldGTPase eluted from the column about halfway through the gradient. Thefirst 90% of the activity peak was pooled, dialyzed versus Buffer I at4° C., and constituted Fraction IV.

[0195] Fraction IV was applied to a prepacked Mono S column (BioRad,Inc.; column volume 1.0 mL) that had been equilibrated with Buffer I.The enzyme was eluted from the column with a 50 mL concave gradient fromzero to 2.0 M KCl at a flow rate of 2.0 mL/min. The enzyme eluted fromthe column during the first quarter of the gradient. The central 95% ofthe activity peak was pooled and constituted Fraction V.

[0196] The final purification step consisted of applying Fraction VdGTPase (without prior dialysis) to a 90 cm×4.9 cm² Sephacryl 200-HRsize exclusion column in Buffer I. The entire eluting dGTPase peak (asvisualized by SDS-PAGE) was pooled and constituted homogeneous, FractionVI enzyme. The enzyme was concentrated via pressure filtration through asemipermeable membrane (Amicon YM 3) and dialyzed into new bufferconstituents as needed. All subsequent investigations were performedwith Fraction VI protein.

[0197] Recombinant Enzyme Expression

[0198] Expression was performed with the T7 RNA polymeraseover-expression system from Novagen, Inc., using the pET11d vector. A 1%innoculum of BL21(DE3)-pLysS cells containing expression plasmidconstructs (pEdgte for E. coli, and pSdgte for S. marcescens) was grownat 37° C. in Luria broth supplemented with 60 mg/mL ampicillin. IPTG wasadded to a final concentration of 0.5 mM when the cells had reached anA595 value of 0.8 (in approximately three hours post inoculation). Cellgrowth continued for five additional hours before harvesting. Enzymeswere purified as described above.

[0199] Production of Polyclonal Antibodies

[0200] Anti-dGTPase polyclonal antibodies (pAb) were produced againsteither Escherichia or Serratia dGTPase. Neutralizing anti dGTPaseantibodies were induced by subcutaneous injection of homogeneous dGTPase(300 mg) in a 1:1 homogenate with Freund″s complete adjuvant into femaleNew Zealand White rabbits. Three subsequent injections of antigen (200mg) with incomplete adjuvant were performed at weekly intervals. Oneweek after the last injection, the rabbits were bled via an ear cannula.The cleared plasma was collected by centrifugation at 14,000×g andstored at 4° C. until use.

[0201] Purification of Polyclonal Antibodies

[0202] The pAbs were purified to homogeneity by affinity chromatographyon DEAE Affi-gel Blue. A BioRad Labs, Inc. rabbit polyclonal antibodyisolation kit was employed according to the supplied instructions, withseveral minor modifications. The protocol was as follows: The clearedrabbit serum (5 mLs) was passed over an Econo-Pac 10DG desalting column.The pAbs were eluted from the column using the supplied running buffer(0.02 M Tris HCl (pH 8.0), 0.028 M NaCl), and were collected as a singlefraction. The protein concentration was determined using the Bradfordassay.

[0203] The entire serum sample (about 25 mLs) was passed over the columnin 5 mL batches. Between batches, the column was washed with 40 mL ofrunning buffer (two column volumes). The final desalted samples from theindividual column runs were pooled. This pooled sample was applied tothe DEAE Affi-gel Blue as a single load, the column was washed with 5column volumes of running buffer (50 mLs), and the pAb fraction waseluted from the column by the application of 5 column volumes of elutionbuffer (0.025 M Tris HCl (pH 8.0), 0.025 M NaCl). The eluted materialwas collected as 5 mL fractions. Purity of the IgG fraction wasestimated by SDS PAGE. Appropriate fractions were pooled, concentratedto 2 mg/mL by pressure filtration, and stored at −70° C. until needed.The DEAE Affi-gel Blue column was regenerated by washing the column with2 M NaCl, 1.5 M sodium thiocyanate in running buffer (10 columnvolumes), followed by re-equilibration in running buffer. The flow ratefor all chromatography steps was maintained at 1.0 mL/min.

[0204] ELISAs and Western Blot Analysis

[0205] ELISAs were performed according to Kaiser and Pollard, (24) oraccording to Quirk et al. (25). Ten μg of partially purified proteinextract, or 1 μg of purified dGTPase was adsorbed to the surface of a96-well microtiter plate (Immulon 2, Dynatech Labs). After the wellswere blocked with phosphate buffered saline (PBS) supplemented with 10%nonfat dry milk, polyclonal antibodies in blocking buffer were added atvarious dilutions and were allowed to react with the antigen at roomtemperature for one hour. Following three washes in PBS, visualizationwas achieved via a goat anti-rabbit secondary antibody that wasconjugated with horse radish peroxidase (Santa Cruz Biotechnology,Inc.). The secondary antibody was added at a 1:2000 dilution in blockingbuffer and was incubated at room temperature for one hour. After threewashes in PBS, color development was achieved by adding a solutioncontaining 50 mM sodium citrate, 50 mM citric acid, 1 mg/mLo-phenylenediamine, and 0.006% H2O2. After suitable color development(typically 5 to 10 minutes of incubation at room temperature) 50 μL of 2M sulfuric acid was added to stop the reaction and stabilize theproduct. Absorbance was measured at 490 nm using an automatic ELISAplate reader (Molecular Dynamics, Inc.). Western blots were madeaccording to (26) and were used to assay antibody specificity and crossreactivity.

[0206] Conjugation of the pAb Pool to CNBr Activated Agarose

[0207] CNBr activated agarose resin (Sigma Chemical Co.) was preswollenin approximately 200 mLs of 0.001 N HCl at room temperature immediatelyprior to use. The coupling capacity of the resin was 1 g dry resin forevery 10 mg of protein. The purified pAb pool was dialyzed againstcoupling buffer (0.1 M H₃BO₃, 25 mM Na₂B₄O₇, 75 mM NaCl, pH 8.4)overnight at 4° C. The resin was poured into a scintered glass funneland was washed extensively with coupling buffer (approximately 25 mLs ofbuffer per 1 mL of resin). The resin was removed from the filter and wasmixed with the pAb pool. This mixture was incubated with constant gentlerocking at room temperature for 4 hours, followed by an overnightincubation at 4° C. The resin was collected on the glass filter. Theprotein concentration in the saved filtrate was determined via Bradfordassay in order to calculate the coupling yield (usually greater than90%). The immunoaffinity resin was removed from the filter and wasresuspended in 15 mL of 1 M ethanolamine (pH 8.0) in order to blockremaining unreacted CNBr groups. After incubation for two hours at roomtemperature (with gentle constant shaking), the resin was collected onthe glass filter and was extensively washed (usually 10 volumes each)with borate buffer (0.1 M H₃BO₃, 25 mM Na₂B₄O₇, 1 M NaCl; pH 8.4),followed by acetate buffer (75 mM NaCOOCH₃, 1 M NaCl). Non covalentlybound protein was removed with this washing step. Finally, the resin waswashed (usually 10 volumes) with coupling buffer, and was resuspended inan excess volume of Tris buffered saline (TBS), pH 7.6 and was stored at4° C. until use. The immunoaffinity resin was utilized to purify dGTPasefrom other genera of the Enterobacteriaceae as described below.

[0208] Isolation of Enteric dGTPases by Immunoaffinity Chromatography

[0209] Twenty mLs of immunoaffinity resin was utilized to purify entericdGTPase from a 2 liter overnight culture (grown in LB media at 30° C.for 12 hours). A crude protein extract was prepared from this materialas follows. Culture medium was centrifuged at 6,000×g in order to pelletthe bacterial cells. The pellet was resuspended in 40 mLs of 30 mMglycine (pH 7.5) and recentrifuged. The final pellet was resuspended in10 mLs of 30 mM glycine (pH 7.5) and was subjected to three cycles ofsonication using a Branson sonicator equipped with a microtip. Allprocedures were carried out at 4° C. unless otherwise specified. Thesonicated material was centrifuged at 10,000×g for 20 minutes, and thesupernatant was decanted and retained.

[0210] This crude protein extract was heated to 60° C. for 15 minuteswith gentle swirling. During this incubation period a milky whiteprecipitate formed. The material was clarified by centrifugation at10,000×g for 20 minutes. The supernatant was again decanted andretained. The crude extract was mixed with 0.1 volume of 5% streptomycinsulfate, was incubated on ice for 60 minutes, and was centrifuged at12,000×g for 20 minutes. The pellet was triturated with 2 mLs of 30 mMKpi (pH 7.5) with gentle mixing and agitation, and then wasrecentrifuged as before. The final supernatant was dialyzed overnight at4° C. against 30 mM Kpi (pH 7.5). This material was then heated to 60°C. with gentle swirling for 15 minutes, was centrifuged at 10,000×g for20 minutes, and the supernatant was decanted. The protein fraction wasthen combined with the affinity resin preparation, and incubated on icefor eight hours with occasional swirling. The slurry was poured into acolumn. The column was washed with 50 mLs of 20 mM glycine (pH 7.5),followed by 20 mLs of 20 mM glycine (pH 7.5), 3 M potassium thiocyanateat a flow rate of 10 mLs per hour. Eluted protein was collected as asingle fraction and immediately concentrated to a volume of 0.1 mL bypressure filtration through a semi permeable membrane (Amicon). Thefinal sample was then extensively dialyzed versus 20 mM KPi (pH 7.5) at4° C. Several such rounds of purification were performed in order tohave sufficient material for studies.

[0211] Biosensor Chip Design

[0212] A CM5 unmodified biosensor chip (27) was obtained from BiaCore,Inc. This chip was a 1 cm square quartz crystal that was coated with athin gold layer. Attached to the gold layer was a tether ofcarboxymethyldextran. The terminal carboxyl groups are easily modifiedin order to create reactive groups for protein coupling. The linkagereaction began with the activation of the chip tether. The chip wasreacted with a solution of 100 mM N-ethyl-N′(dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mM N-hydroxysuccinimide (NHS) in25 mM sodium bicarbonate (pH 8.5) for 5 minutes, followed by a briefrinse with 100 mM borate (pH 8.5). The activated CM5 chip was incubatedin 80 mM 2-(2-pyridinyldithio)ethaneamine (PDEA), 0.1 M borate (pH 8.5)for four minutes, followed by a brief rinse in 0.1 M borate (pH 8.5).The chip was then placed in contact with a solution (200 μl) containingpurified pAb at a concentration of 0.05 mg/mL for 10 minutes. Finally,all reactive disulfides were deactivated, and non-covalently boundprotein was removed by soaking the chip surface in 200 μL of 50 mMcysteine, 1 M NaCl for 10 minutes. All reactions were done at roomtemperature. The final biosensor was used in a BiaCore surface plasmoninstrument according to the instructions from the manufacturer. Flowrate over the sensor surface was 50 μL per minute.

EXAMPLE 2 Immunological Relationship Between Enterobacteriaceae dGTPases

[0213] The immunological relatedness of dGTPase among severalEnterobacteriaceae genera was examined by using purified anti-dGTPasepolyclonal antibodies (pAbs) combined with an enzymatic assay for theprotein. This approach was highly sensitive and specific for dGTPaseactivity. Table 5 provides the specific activities and generalizedantibody reactivities of dGTPases from several genera ofEnterobacteriaceae that were partially purified as described in Example1 (Fractions II and III). Immunological reactivities with two separateantibody preparations were observed: anti-Escherichia coli dGTPase andanti-Serratia marcescens dGTPase. TABLE 5 Specific Activity BacterialType (units/mg) pAB Reactivity¹ Bacterial Species ATCC No. Fraction IIFraction III Anti-E Anti-S Cedecca davisae 33431 1.4 20.0 ++ −Citrobacter freundii 8090 0.8 15.0 + − Enterobacter aerogens 13048 0.611.0 − +++ Escherichia coli 25257 1.3 27.0 +++ − Escherichia coliO157:H7 35150 1.2 25.0 +++ − Escherichia coli O111 33780 1.3 26.0 +++ −Escherichia coli O124:NM 43893 1.3 25.8 +++ − Escherichia fergusonii35469 1.2 26.2 +++ − Hafnia alvei 29926 0.5 9.0 − ++ Klebsiella oxytoca43165 2.3 20.6 + − Klebsiella pneumoniae 13883 2.2 20.2 + − Proteusmirabilis 7002 3.1 19.9 − + Proteus vulgaris 13315 2.8 18.6 − ++Salmonella enteritidis 13076 1.5 14.8 ++ − Salmonella gallinarum 91841.7 14.2 ++ − Salmonella typhi 6539 1.2 14.3 ++ − Salmonella typhimurium14028 1.1 14.0 ++ − Serratia marcescens 8100 1.6 17.3 − +++ Serratiaodorifera 33077 1.5 17.0 − +++ Shigella boydii 9207 2.4 19.7 +++ −Shigella dysenteriae 13313 2.3 19.0 +++ − Yersinia enterocolitica 237150.6 8.4 − +++ Yersinia intermedia 29909 0.8 9.1 − +++

[0214] Enzyme specific activities ranging from slightly lower toslightly higher than that observed for the dGTPase from E. coli wereseen. All of the enteric dGTPases exhibit similar fractionation behaviorthroughout the partial purification protocol, suggesting that many ofthe physico-chemical properties of the various dGTPases were similar.However, as indicated in Table 5, the immunological reactivities of thevarious dGTPases were not identical.

[0215] Moreover, an antibody raised against one type of dGTPase couldinhibit enzyme activity of dGTPases from only a subset ofEnterobacteriaceae genera. Polyclonal antibodies produced against eitherthe E. coli or the S. marcescens enzyme were tested for their ability toinhibit dGTPase activity among various bacteria. As illustrated in FIGS.1-4, the ability of an antibody to inhibit dGTPase activity wasdependent on the source of antigen used to generate the antibody. Thedependence of antibody inhibition upon the concentration of pAb is alsoillustrated in FIGS. 1-4.

[0216]FIG. 1 shows the effect of anti-E. coli dGTPase polyclonalantibodies upon enzymatic activity of dGTPases isolated from Y.enterocolitica (open circles), E. aerogens (closed triangles), P.vulgaris (open diamonds) K. oxytoca (open triangles), S. typhimurium(open squares), S. boydii (closed circles), and C. davisae. Enzyme(Fraction III) was incubated with various amounts of pAb for 15 minutesat 25° C., followed by measurement of residual enzyme activity using aradioactive assay for the production of tripolyphosphate (16). Percentactivity of a control enzyme preparation (no added pAb) was arbitrarilyset to 100 percent. Three of the tested enteric genera, Salmonella,Shigella, and Cedecca were effectively inhibited by anti-EscherichiapAbs. Three of the tested genera, Proteus, Yersinia and Enterobacter,were not inhibited by the addition of the pAb preparation. Klebsiellawas inhibited by the addition of anti-Escherichia pAbs, but only half aseffectively as the Salmonella, Shigella, and Cedecca genera.

[0217] The ability of anti-Serratia marcescens dGTPase pAbs to inhibitthe enzymatic activity of these dGTPases was assayed as above and ispresented in FIG. 2. The enzyme activities of dGTPases isolated from S.typhimurium (open squares), K. oxytoca (open triangles), S. boydii(closed circles), P. vulgaris (open diamonds), Y. enterocolitica (opencircles), H. alvei (closed diamonds), E. aerogens (closed triangles),and S. marcescens (closed squares) in the presence of anti-Serratiamarcescens dGTPase is shown in FIG. 2. Anti-Serratia marcescens dGTPasepAbs do not significantly alter the enzymatic activity of Klebsiella,Salmonella, or Shigella genera. These antibodies were, however,effective in inhibiting the enzymatic activity of Enterobacter,Yersinia, and to a lesser extent, Proteus. The loss of enzymaticactivity in the presence of pAb therefore provides an assay for thefunctional relatedness or cross reactivity of dGTPases from differentspecies. It is also a relatively rapid assay with a sensitivitycomparable to that of an ELISA analysis.

[0218] Further immunological cross reactivity was assayed by ELISA asshown in FIG. 3. The reactivity of anti-Escherichia dGTPase pAbs withimmobilized dGTPases from E. coli 0157 (closed circles), S. boydii (opencircles), S. typhimurium (closed squares), K. oxytoca (closedtriangles), E. aerogens (closed diamonds), C. davisae (open squares), Y.enterocolitica (open diamonds), and C. freundii (open triangles) wasobserved by reacting a secondary antibody with any bound pAb.Anti-Escherichia dGTPase pAbs efficiently bind enzymes from Shigella,Cedecca, Salmonella, Klebsiella, and to a lesser extent, Citrobacter.The pAbs also bind to dGTPases from other Escherichia species. The ELISAassay shows no reactivity towards Enterobacter or Yersinia dGTPases.

[0219] Anti-Serratia dGTPase pAbs binding to various dGTPases by ELISAassay is shown in FIG. 4. Fraction III enzyme preparation from S.typhimurium (closed triangles), E. coli (open triangles), H. alvei (opensquares), E. aerogens (closed circles), Y. enterocolitica (opencircles), and P. vulgaris (closed squares) was adsorbed onto microtiterplates and the amount of anti-Serratia dGTPase pAbs bound was detectedwith a secondary antibody. The anti-Serratia dGTPase antibodypreparation efficiently detects antigen from Enterobacter, Yersinia,Proteus, and Hafnia. dGTPases from Salmonella and Escherichia were notdetected with these anti-Serratia pAbs. These ELISA results were incomplete accord with the loss of enzymatic activity shown in FIGS. 1 and2, and summarized in Table 5.

[0220] Significantly, by using only antibody preparations fromEscherichia coli and Serratia marcescens, it was possible to detectdGTPases from all major enteric genera. Moreover, by using just thesetwo antibody preparations, it was possible to differentiate between thetypes of bacteria detected. Such differentiation can help identify whichgenus of bacteria is present in a test sample.

[0221] Anti-Serratia or anti-Escherichia immunoaffinity columns wereemployed to isolate nearly homogenous dGTPase preparations from nineenteric bacteria. FIG. 5A provides a SDS PAGE analysis of theimmunoaffinity column-purified enzymes. Immunopurified dGTPases fromnine species were all capable of hydrolyzing dGTP to deoxyguanosine andtripolyphosphate. They were all thermostable. All isolated dGTPases,with the exception of Serratia dGTPase, bind to single-stranded DNA.However, while most dGTPases are tetrameric, Serratia dGTPases aredimeric. These results are summarized in Table 6. TABLE 6 dGTPaseStability Structure ssDNA Binding Km kcat t{fraction (1/2 )} at 65° C.Quaternary Ka Stimulation Bacterial Species (μM) (s⁻¹) (min) structure(M⁻¹ × 10⁶) (fold) C. davisae 7.9 3379 16.8 tetramer 5.3 1.5 E. aerogens5.6 3720 23.0 tetramer 5.6 1.5 E. coli O157.H7 6.2 4032 21.5 tetramer6.3 1.7 E. coli 6.0 4020 22.0 tetramer 6.2 1.6 H. alvei 9.1 3655 17.5tetramer 4.7 1.4 K. pneumoniae 6.0 4002 23.0 tetramer 6.0 1.5 P.vulgaris 3.7 4112 31.5 tetramer 7.1 1.9 S. enteritidis 6.2 3218 23.3tetramer 6.1 1.6 S. marcescens 3.0 3975 18.7 dimer nd* 1.0 Y.enterocolitica 5.8 3982 27.0 tetramer 7.0 1.8

[0222]FIG. 5B shows that the anti-Serratia and anti-Escherichia dGTPaseantibodies used in these studies were specific for the detection of thedGTPase enzyme, and do not cross react with other bacterial proteins incrude extracts. Only a single band was observed on the Western blotshown in FIG. 5B, indicating that the anti-Serratia pAbs only detectedSerratia dGTPase and the anti-Escherichia pAbs only detected EscherichiadGTPase. No cross-reactivity was observed. This observation is furthersupported by the enzymatic studies, where the anti-Serratia pAbs did notappreciably bind to the Escherichia dGTPase and did not inhibitenzymatic activity of dGTPases from genera related to Escherichia, andvice versa. Such immuno-specificity is highly desirable for constructionof an accurate diagnostic test for enteric bacterial contamination.

EXAMPLE 3 Biosensor Chips for Detection of Enterobacteriaceae

[0223] Biosensor chips were made and utilized for easy detection ofenteric dGTPases. Using the induced thiol coupling method described inExample 1, the surface of a CM5 chip was modified by attaching eitheranti-Escherichia or anti-Serratia dGTPase pAbs. These biosensors wereemployed for the detection of enteric dGTPases using a BiaCore 2000surface plasmon resonance (SPR) device.

[0224] The results of these experiments are shown in FIGS. 6 and 7. FIG.6 provides a surface plasmon resonance (SPR) sensogram showing thereactivity of tethered anti-E. coli dGTPase pAbs with crude bacterialextracts from E. coli (Ec), S. boydii (Sb), C. daviseae (Cd), K. oxytoca(Ko), S. typhimurium (St), C. freundii (Cf), and E. aerogens (Ea). Asshown in FIG. 6, anti-E. coli dGTPase pAbs react most strongly with E.coli (Ec), S. boydii (Sb), S. typhimurium (St), and C. daviseae (Cd).

[0225]FIG. 7 provides a surface plasmon resonance (SPR) sensogramshowing the reactivity of tethered anti-Serratia marcescens dGTPase pAbswith crude bacterial extracts from S. marcescens (Sm), E. aerogens (Ea),Y. enterocolitica (Ye), P. vulgaris (Pv), E. coli (Ec), and S.typhimurium (St). As shown in FIG. 7, anti-Serratia marcescens dGTPasepAbs react most strongly with S. marcescens (Sm), E. aerogens (Ea), Y.enterocolitica (Ye), and P. vulgaris (Pv).

[0226] The sensograms shown in FIGS. 6 and 7 are highly reproduciblewith standard deviations of +1% of signal intensity across the bindingisotherm. The biosensor chips have been reused up to 10 times withoutdegradation of the SPR signal.

[0227] Both biosensors chips have the ability to detect dGTPase in crudebacterial extract and they can discriminate between types of dGTPases.The binding interaction affinity observed in the SPR system mirrors thatobserved using the ELISA assay. In addition the biosensors candifferentially detect bacterial genera as shown by the sensogram bindingisotherms. There was nearly no detectable background binding (even usingcrude protein extract) as evidenced by the nearly flat binding isothermfor a gram positive, non enteric bacterium (S. aureus, marked Sa in FIG.6.

[0228] The chips used above were titrated with increasing amounts ofdGTPase in order to determine the detection limit in the system. Theresults of this experiment are shown in FIG. 8. The chip with theanti-Escherichia pAbs was able to detect 1 μg of purified E. colidGTPase. A similar detection limit was observed when the biosensor chiphaving anti-Serratia pAbs when it was reacted with purified SerratiadGTPase.

[0229] Hence, the polyclonal antibody preparations made as describedherein were capable of specifically detecting low amounts of enzymewithin the background of other bacterial proteins, all within 400seconds. Such differential reactivities indicate not only thatEnterobacteriaceae can be detected in complex test samples but that thetype of Enterobacteriaceae can be identified.

EXAMPLE 4 PCR Detection and Identification of Enterobacteriaceae

[0230] The invention provides nucleic acids that can be used to detectEnterobacteriaceae and to identify which genus of Enterobacteriaceae ispresent in a sample. In this example, the nucleic acids are used in aseries of polymerase chain reaction (PCR) amplification experiments todemonstrate the specificity and sensitivity of the present detection andidentification methods.

[0231] Using the DNA sequence of the E. coli gene (SEQ ID NO:1, FIG. 9),PCR primers were identified and synthesized that could amplify anyenteric dgt gene (Table 7). Separate PCR primers were identified andsynthesized that could specifically amplify dgt sequences from a singleenteric genus (Table 7a-7c). The primers were used in pairs that werelabeled primer pairs 1 to 13, as shown in Tables 6 and 7a-7c. TABLE 7Oligonucleotide primer pairs to amplify any enteric dgt gene sequenceAmplified DNA Pair SEQ ID Position in Length No. Sequence NO: SEQ IDNO:1 (bp) 1 CCACTGGAGCGCAATG 2 166-181 184 1 TGCATCAGGCATGACAT 3 334-350184 2 CCACTGGAGCGCAATG 2 166-181 215 2 AAAATGACCAAACGGCGG 4 364-381 2153 GGGCGCTACATCGC 5 229-242 121 3 TGCATCAGGCATGACAT 3 334-350 121 4GGGCGCTACATCGC 5 229-242 152 4 AATGACCAAACGGCGG 6 364-381 152

[0232] TABLE 8a Oligonucleotide primer pairs to specifically amplifyEscherichia coil dgt nucleic acids Amplified DNA Pair SEQ ID Position inLength No. Sequence NO: SEQ ID NO:1 (bp) 5 GCTGCAGCGTGGCGGCA 7 461-477213 5 CTCAGGCGTTTCGCCACG 8 656-673 213 6 CCCGGAAGATGCCGAAA 9 423-439 2516 CTCAGGCGTTTCGCCACG 8 656-673 251 7 CCCGGAAGATGCCGAAA 9 423-439 82 7CGGTTCTTCCCCGTCCC 10 488-504 82

[0233] TABLE 8b Oligonucleotide primer pairs to specifically amplifySalmonella typhymurium nucleic acids Amplified DNA Pair SEQ ID Positionin Length No. Sequence NO: SEQ ID NO:1 (bp) 8 GCTGTGTGGTTTCCTCG 11461-477 213 8 AATCCGGCACCGGCCCTC 12 656-673 213 9 TCCGGAAGATGCGGAAA 13423-439 251 9 AATCCGGCACCGGCCCTC 12 656-673 251 10 TCCGGAAGATGCGGAAA 13423-439 82 10 ATTTTCTTCACCTTCCT 14 488-504 82

[0234] TABLE 8c Oligonucleotide primer pairs to amplify Klebsiellaoxytoca nucleic acids Amplified DNA Pair SEQ ID Position in Length No.Sequence NO: SEQ ID NO:1 (bp) 11 GCTGCGAAGTGCAGGCC 15 461-477 213 11TGGCCGGCGTCTCCTCAG 16 656-673 213 12 GCCCGGCGATGCGCTCG 17 423-439 251 12TGGCCGGCGTCTCCTCAG 16 656-673 251 13 GCCCGGCGATGCGCTCG 17 423-439 82 13CGATGTCTCTCCGTCAT 18 488-504 82

[0235] Polymerase chain reaction (PCR) amplification of DNA wasperformed using VENT thermopolymerase from New England Biolabs, Inc.according to the instructions provided by the manufacturer. All PCRreactions were carried out in a ProGene thermocycler from Techne, Inc.In general, the amplification products were visualized after sizeseparation on a 1.2% agarose gel by staining with ethidium bromide.

[0236]FIG. 10 illustrates that the methods of the invention can detectas few as 10 to 100 Enterobacteriaceae in a test sample. Primer set 1was used to amplify a 184 bp fragment from approximately 1000 E. colibacteria in lane 1, from approximately 100 E. coli bacteria in lane 2,and from approximately 10 E. coli bacteria in lane3. Only thirty cyclesof the PCR reaction were performed. The band in lane 3 is just visiblein the original gel, where only about 10 copies of template DNA werepresent.

[0237] The primers provided in Tables 7a-7c are specific for theindicated genus of Enterobacteriaceae. FIG. 11 illustrates that primerset 5, that was designed to be specific for Escherichia, only amplifiesDNA isolated from Escherichia. No DNA amplification was observed whenthe template DNA was from Klebsiella, Salmonella, Shigella or Yersinia.

[0238] Moreover, the primers provided in Tables 7a-7c can stilldiscriminate between DNA substrates, even when mixtures of DNA and morethan one set of primers is present in the amplification reaction. Asillustrated in FIGS. 12 and 13, designing the genus-specific primer setsso that bands of different lengths are amplified by each is one way tofacilitate identification of the specific bacterial types in mixedcultures.

[0239]FIG. 12 shows that the genus-specific primers provided by theinvention can discriminate between different types of EnterobacteriaceaeDNA under conditions where different primers are present and may competefor primer binding and amplification. Mixtures of DNA obtained fromapproximately 200 cfu of two species of Enterobacteriaceae wereamplified. The products were separated on a 1.2% agarose gel and stainedwith ethidium bromide. The species of bacterial DNA present in thesamples and the primers used to test the specificity of amplificationare provided below. Lane Bacteria Primer Pair Band size (bp) 1Escherichia + Klebsiella 7 and 11 82 (Escherichia) + 213 (Klebsiella) 2Escherichia + Salmonella 7 and 8 82 (Escherichia) + 213 (Salmonella) 3Salmonella + Klebsiella 8 and 13 213 (Salmonella) + 82 (Klebsiella) 4Klebsiella + Escherichia 6 251 (Escherichia) 5 Salmonella + Escherichia6 251 (Escherichia)

[0240] As shown in Tables 7a-7c, primer pairs 5, 6 and 7 were designedto be specific for Escherichia; primer pairs 8, 9 and 10 were designedto be specific for Salmonella; primer pairs 11, 12 and 13 were designedto be specific for Klebsiella. Each of the primer pairs employedactually synthesized a fragment of the predicted size for a particulargenus of bacteria. Thus, the methods of the invention discriminatebetween DNA from related Enterobacteriaceae and correctly identify whichbacterial genus is present in a mixed bacterial culture. FIG. 12 showsthat the primer sets can be used in combination to amplify dgt sequencefrom bacteria in mixed cultures.

[0241] When multiple bands were visualized (as in FIG. 12, lane 1 forexample) it is straightforward to make the identification. Finally thePCR based test can be used to amplify dgt genetic material from fieldsamples. FIG. 13 shows the results from a series of amplifications usingmultiple primers. The sample was the fluid found in the bottom of asealed package of commercial chicken. The results clearly indicate thatthe fluid contains measurable amounts of enteric bacteria (lanes 1 and7), that it is Escherichia (lanes 2 and 6), not Klebsiella (lane 3), andthat it is Salmonella (lanes 4 and 5). This result also indicates thatthe PCR based test does not amplify spurious materials as evidenced bythe lack of extra bands in the individual lanes, and that the test isnot inhibited by serum or other components in the fluid.

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[0283] 42. Janda, J. M., and Abbott, S. L. The Enterobacteria.Lippincott-Raven, Philadelphia 1998.

[0284]

1 36 1 1518 DNA Escherichia coli 1 atggcacaga ttgatttccg aaaaaaaataaactggcatc gtcgttaccg ttcaccgcag 60 ggcgttaaaa ccgaacatga gatcctgcggatcttcgaga gcgatcgcgg gcgtatcatc 120 aactctccgg caattcgtcg tctgcaacaaaagacccagg tttttccact ggagcgcaat 180 gccgccgtgc gcacgcgtct tacccactcgatggaagtcc agcaggtggg gcgctacatc 240 gccaaagaaa ttttaagccg tctgaaagagcttaaattac tggaagcata cggcctggat 300 gaactgaccg gtccctttga aagcattgttgagatgtcat gcctgatgca cgatatcggc 360 aatccgccgt ttggtcattt tggcgaagcggcgataaatg actggtttcg ccaacgtttg 420 cacccggaag atgccgaaag ccagcctctgactgacgatc gctgcagcgt ggcggcacta 480 cgtttacggg acggggaaga accgcttaacgagctgcggc gcaagattcg tcaggactta 540 tgtcattttg aggggaatgc acaaggcattcgcctggtgc atacattgat gcggatgaat 600 ctcacctggg cacaggttgg cggtattttaaaatataccc gtccggcgtg gtggcgtggc 660 gaaacgcctg agacacatca ctatttaatgaaaaagccgg gttattatct ttctgaagaa 720 gcctatattg cccggttgcg taaagaacttaatttggcgc tttacagtcg ttttccatta 780 acgtggatta tggaagctgc cgacgacatctcctattgtg tggcagacct tgaagatgcg 840 gtagagaaaa gaatatttac cgttgagcagctttatcatc atttgcacga agcgtggggc 900 cagcatgaga aaggttcgct cttttcgctggtggttgaaa atgcctggga aaaatcacgc 960 tcaaatagtt taagccgcag tacggaagatcagtttttta tgtatttacg ggtaaacacc 1020 ctaaataaac tggtacccta cgcggcacaacgatttattg ataatctgcc tgcgattttc 1080 gccggaacgt ttaatcatgc attattggaagatgccagcg aatgcagcga tcttcttaag 1140 ctatataaaa atgtcgctgt aaaacatgtgtttagccatc cagatgtcga gcggcttgaa 1200 ttgcagggct atcgggtcat tagcggattattagagattt atcgtccttt attaagcctg 1260 tcgttatcag actttactga actggtagaaaaagaacggg tgaaacgttt ccctattgaa 1320 tcgcgcttat tccacaaact ctcgacgccgcatcggctgg cctatgtcga ggctgtcagt 1380 aaattaccgt cagattctcc tgagtttccgctatgggaat attattaccg ttgccgcctg 1440 ctgcaggatt atatcagcgg tatgaccgacctctatgcgt gggatgaata ccgacgtctg 1500 atggccgtag aacaataa 1518 2 16 DNAArtificial Sequence A primer. 2 ccactggagc gcaatg 16 3 17 DNA ArtificialSequence A primer. 3 tgcatcaggc atgacat 17 4 18 DNA Artificial SequenceA primer. 4 aaaatgacca aacggcgg 18 5 14 DNA Artificial Sequence Aprimer. 5 gggcgctaca tcgc 14 6 16 DNA Artificial Sequence A primer. 6aatgaccaaa cggcgg 16 7 17 DNA Artificial Sequence A primer. 7 gctgcagcgtggcggca 17 8 18 DNA Artificial Sequence A primer. 8 ctcaggcgtt tcgccacg18 9 17 DNA Artificial Sequence A primer. 9 cccggaagat gccgaaa 17 10 17DNA Artificial Sequence A primer. 10 cggttcttcc ccgtccc 17 11 17 DNAArtificial Sequence A primer. 11 gctgtgtggt ttcctcg 17 12 18 DNAArtificial Sequence A primer. 12 aatccggcac cggccctc 18 13 17 DNAArtificial Sequence A primer. 13 tccggaagat gcggaaa 17 14 17 DNAArtificial Sequence A primer. 14 attttcttca ccttcct 17 15 17 DNAArtificial Sequence A primer. 15 gctgcgaagt gcaggcc 17 16 18 DNAArtificial Sequence A primer. 16 tggccggcgt ctcctcag 18 17 17 DNAArtificial Sequence A primer. 17 gcccggcgat gcgctcg 17 18 17 DNAArtificial Sequence A primer. 18 cgatgtctct ccgtcat 17 19 496 PRTEscherichia coli 19 Met Ala Gln Ile Asp Phe Arg Lys Lys Ile Asn Trp HisArg Arg Tyr 1 5 10 15 Arg Ser Pro Gln Gly Val Lys Thr Glu His Glu IleLeu Arg Ile Phe 20 25 30 Glu Ser Asp Arg Gly Arg Ile Ile Asn Ser Pro AlaIle Arg Arg Leu 35 40 45 Gln Gln Lys Thr Gln Val Phe Pro Leu Glu Arg AsnAla Ala Val Arg 50 55 60 Thr Arg Leu Thr His Ser Met Glu Val Gln Gln ValGly Arg Tyr Ile 65 70 75 80 Ala Lys Glu Ile Leu Ser Arg Leu Lys Ser LeuAsn Thr Glu Leu Thr 85 90 95 Gly Pro Phe Glu Ser Ile Val Glu Tyr Ala CysLeu Met His Asp Ile 100 105 110 Ala Ile Arg Arg Leu Val Ile Leu Ala LysArg Thr Ile Asn Asp Trp 115 120 125 Phe Gly Gln Arg Leu His Pro Glu AspAla Glu Ser Gln Pro Leu Thr 130 135 140 Asp Arg Cys Ser Val Ala Ala LeuArg Leu Arg Thr Gly Lys Asn Arg 145 150 155 160 Leu Thr Ser Cys Gly AlaArg Phe Val Arg Thr Tyr Val Ile Leu Arg 165 170 175 Gly Met His Lys HisSer Pro Gly Ala Tyr Ile Asp Ala Asp Glu Ser 180 185 190 His Leu Gly ThrGly Trp Arg Tyr Phe Lys Ile Tyr Pro Ser Gly Val 195 200 205 Val Ala CysGlu Thr Pro Glu Thr His His Tyr Leu Met Lys Lys Pro 210 215 220 Gly TyrTyr Leu Ser Glu Glu Ala Tyr Ile Ala Arg Leu Arg Lys Glu 225 230 235 240Leu Asn Leu Ala Leu Tyr Ser Arg Phe Pro Leu Thr Trp Ile Met Glu 245 250255 Ala Ala Asp Asp Ile Ser Tyr Cys Val Ala Asp Leu Glu Asp Ala Val 260265 270 Glu Lys Arg Ile Phe Thr Val Glu Gln Leu Tyr His His Leu His Glu275 280 285 Ala Trp Gly Gln His Glu Lys Gly Ser Leu Phe Ser Leu Val ValGlu 290 295 300 Asn Ala Trp Glu Lys Ser Arg Ser Asn Ser Leu Ser Arg SerThr Glu 305 310 315 320 Asp Gln Phe Phe Met Tyr Leu Arg Val Asn Thr LeuAsn Lys Leu Val 325 330 335 Pro Tyr Ala Ala Gln Arg Phe Ile Asp Asn LeuPro Ala Ile Phe Ala 340 345 350 Gly Arg Phe Asn His Ala Leu Leu Glu AspAla Ser Glu Cys Ser Asp 355 360 365 Leu Leu Lys Leu Tyr Lys Asn Val AlaVal Lys His Val Phe Ser His 370 375 380 Pro Asp Val Glu Arg Leu Glu LeuGln Gly Tyr Arg Val Ile Ser Gly 385 390 395 400 Leu Leu Glu Ile Tyr ArgPro Leu Leu Ser Leu Ser Leu Ser Asp Phe 405 410 415 Thr Glu Leu Val GluLys Glu Arg Val Lys Arg Phe Pro Ile Glu Ser 420 425 430 Arg Leu Phe HisLys Leu Ser Thr Pro His Arg Leu Ala Tyr Val Glu 435 440 445 Ala Val SerLys Leu Pro Ser Asp Ser Pro Glu Phe Pro Leu Trp Glu 450 455 460 Tyr TyrTyr Arg Cys Arg Leu Leu Gln Asp Tyr Ile Ser Gly Met Thr 465 470 475 480Asp Leu Tyr Ala Trp Asp Glu Tyr Arg Arg Leu Met Ala Val Glu Gln 485 490495 20 495 PRT Salmonella typhimurium 20 Met Ala Ser Ile Asp Phe Arg AsnLys Ile Asn Trp His Arg Arg Tyr 1 5 10 15 Arg Ser Pro Gln Gly Val LysThr Glu His Glu Ile Leu Arg Ile Phe 20 25 30 Glu Ser Asp Arg Gly Arg LeuIle Asn Ser Pro Ala Ile Arg Arg Leu 35 40 45 Gln Gln Lys Thr Gln Val PhePro Leu Glu Arg Asn Ala Ala Val Arg 50 55 60 Thr Arg Leu Thr His Ser MetGlu Val Gln Gln Val Gly Arg Tyr Ile 65 70 75 80 Ala Lys Glu Ile Leu SerArg Leu Lys Glu Gln Asp Arg Leu Glu Glu 85 90 95 Tyr Gly Leu Asp Ala LeuThr Gly Pro Phe Glu Ser Ile Val Glu Met 100 105 110 Ala Cys Leu Met HisAsp Ile Gly Asn Pro Pro Phe Gly His Phe Gly 115 120 125 Glu Ala Ala IleAsn Asp Trp Phe Arg Gln Arg Leu His Pro Glu Asp 130 135 140 Ala Glu SerGln Pro Leu Thr His Asp Arg Cys Val Val Phe Ser Leu 145 150 155 160 ArgLeu Gln Lys Tyr Val Arg Asp Ile Cys His Leu Lys Ala Cys Thr 165 170 175Arg Glu Phe Val Cys Thr Ile Arg Ser Cys Gly Gly Ile Leu Thr Trp 180 185190 Ala Ala Val Arg Pro Asn Phe Lys Asn Ile Pro Val Pro Ala Cys Trp 195200 205 Pro Arg Gly Arg Ser Arg Ile Pro Ile Arg Tyr Leu Met Lys Lys Pro210 215 220 Arg Tyr Tyr Leu Ser Glu Glu Lys Tyr Ile Ala Arg Leu Arg LysGlu 225 230 235 240 Leu Gln Leu Arg Pro Tyr Ser Arg Phe Pro Leu Thr TrpIle Met Glu 245 250 255 Ala Ala Asp Asp Ile Ser Tyr Cys Val Ala Asp LeuGlu Asp Ala Val 260 265 270 Glu Lys Arg Ile Phe Ser Val Glu Gln Leu TyrHis His Leu Tyr His 275 280 285 Ala Trp Cys His His Glu Lys Asp Ser LeuPhe Glu Leu Val Val Gly 290 295 300 Asn Ala Trp Glu Lys Ser Arg Ala AsnThr Leu Ser Arg Ser Thr Glu 305 310 315 320 Asp Gln Phe Phe Met Tyr LeuArg Val Asn Thr Leu Asn Lys Leu Val 325 330 335 Pro Tyr Ala Gln Arg PheIle Asp Asn Leu Pro Gln Ile Phe Ala Gly 340 345 350 Thr Phe Asn Gln AlaLeu Leu Glu Asp Ala Ser Gly Phe Ser Arg Leu 355 360 365 Leu Glu Leu TyrLys Asn Val Ala Val Glu His Val Phe Ser His Pro 370 375 380 Asp Val GluGln Leu Glu Leu Gln Gly Tyr Arg Val Ile Ser Gly Leu 385 390 395 400 LeuAsp Ile Tyr Gln Pro Leu Leu Ser Leu Ser Leu Asn Asp Phe Arg 405 410 415Glu Leu Val Glu Lys Glu Arg Leu Lys Arg Phe Pro Ile Glu Ser Arg 420 425430 Leu Phe Gln Lys Leu Ser Thr Arg His Arg Leu Ala Tyr Val Glu Val 435440 445 Val Ser Lys Leu Pro Thr Asp Ser Ala Glu Tyr Pro Val Leu Glu Tyr450 455 460 Tyr Tyr Arg Cys Arg Leu Ile Gln Asp Tyr Ile Ser Gly Met ThrAsp 465 470 475 480 Leu Tyr Ala Trp Asp Glu Tyr Arg Arg Leu Met Ala ValGlu Gln 485 490 495 21 332 PRT Klebsiella oxytoca 21 Met Ala Lys Ile AspPhe Arg Asn Lys Ile Asn Trp Arg Arg Arg Phe 1 5 10 15 Arg Ser Pro ProArg Val Glu Thr Glu Arg Asp Ile Leu Arg Ile Phe 20 25 30 Glu Ser Asp ArgGly Arg Ile Val Asn Ser Pro Ala Ile Arg Arg Leu 35 40 45 Gln Gln Lys ThrGln Val Phe Pro Leu Glu Arg Asn Gly Arg Val Arg 50 55 60 Thr Arg Leu ThrHis Ser Leu Glu Val Gln Gln Val Gly Arg Tyr Ile 65 70 75 80 Ala Lys GluVal Leu Ser Arg Leu Lys Glu Leu Arg Leu Leu Glu Glu 85 90 95 Tyr Gly LeuGlu Glu Leu Thr Gly Pro Phe Glu Ser Val Val Glu Met 100 105 110 Ala CysLeu Met His Asp Ile Gly Asn Pro Pro Phe Gly His Phe Gly 115 120 125 GluAla Ala Ile Asn Asp Trp Phe Arg Gln Arg Leu Ala Pro Gly Asp 130 135 140Ala Leu Gly Gln Pro Leu Thr Asp Asp Arg Cys Glu Val Gln Ala Leu 145 150155 160 Arg Leu His Asp Gly Glu Thr Ser Leu Asn Ala Leu Arg Arg Lys Val165 170 175 Arg Gln Asp Leu Cys Ser Phe Glu Gly Asn Ala Gln Gly Ile ArgLeu 180 185 190 Val His Thr Leu Met Arg Met Asn Leu Thr Trp Ala Gln ValGly Cys 195 200 205 Ile Leu Lys Tyr Thr Arg Pro Ala Trp Trp Ser Glu GluThr Pro Ala 210 215 220 Ser His Ser Tyr Leu Met Lys Lys Pro Gly Tyr TyrLeu Ala Glu Glu 225 230 235 240 Glu Tyr Val Ala Arg Leu Arg Lys Glu LeuAsp Leu Ala Pro Tyr Asn 245 250 255 Arg Phe Pro Leu Thr Trp Ile Met GluAla Ala Asp Asp Ile Ser Tyr 260 265 270 Cys Val Ala Asp Leu Glu Asp AlaVal Glu Lys Arg Ile Phe Ser Ala 275 280 285 Glu Gln Leu Tyr Gln His LeuTyr Asp Ala Trp Gly Ser His Val Lys 290 295 300 Arg Ser Arg Tyr Ser GlnVal Val Glu Asn Ala Trp Glu Lys Ser Arg 305 310 315 320 Ala Asn Tyr LeuLys Gln Ser Ala Glu Asp Gln Phe 325 330 22 7 PRT Salmonella 22 His ProAsp Glu Ala Glu Ser 1 5 23 7 PRT Klebsiella 23 Ala Pro Gly Asp Ala LeuGly 1 5 24 7 PRT Yersinia 24 Asp Pro Asn Gly Gly Gly Ala 1 5 25 4 PRTSalmonella 25 Val Val Phe Ser 1 26 4 PRT Escherichia 26 Ser Val Ala Ala1 27 4 PRT Klebsiella 27 Glu Val Gln Ala 1 28 4 PRT Yersinia 28 Leu ValAsn Thr 1 29 9 PRT Salmonella 29 Gln Glu Gly Glu Glu Asn Leu Asn Asp 1 530 9 PRT Escherichia 30 Arg Asp Gly Glu Glu Pro Leu Asn Glu 1 5 31 9 PRTKlebsiella 31 His Asp Gly Glu Thr Ser Leu Asn Ala 1 5 32 9 PRT Yersinia32 Arg Glu Gly Glu Thr Glu Leu Asn Ile 1 5 33 7 PRT Salmonella 33 ArgSer Arg Ile Pro Ile Arg 1 5 34 7 PRT Escherichia 34 Glu Thr Pro Glu ThrHis His 1 5 35 7 PRT Klebsiella 35 Glu Thr Pro Ala Ser His Ser 1 5 36 7PRT Yersinia 36 Asp Ile Pro Thr Ser His Asn 1 5

What is claimed:
 1. An isolated nucleic acid that comprises SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ IDNO:18.
 2. An isolated nucleic acid that comprises SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, andthat can selectively hybridize to DNA from a bacteria of the familyEnterobacteriaceae.
 3. An isolated nucleic acid that comprises SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 and that can selectivelyhybridize to DNA from Escherichia coli.
 4. The isolated nucleic acid ofclaim 3 wherein the nucleic acid selectively hybridizes to DNA fromEscherichia coli in the presence of DNA from at least one otherbacterial species of the family Enterobacteriaceae.
 5. The isolatednucleic acid of claim 3 wherein the nucleic acid selectively hybridizesto DNA from Escherichia coli in the presence of DNA from Klebsiella,Salmonella, Shigella or Yersinia.
 6. An isolated nucleic acid thatcomprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 andthat can selectively hybridize to DNA from Salmonella typhymurium. 7.The isolated nucleic acid of claim 6 wherein the nucleic acidselectively hybridizes to DNA from Salmonella typhymurium in thepresence of DNA from at least one other bacterial species of the familyEnterobacteriaceae.
 8. The isolated nucleic acid of claim 3 wherein thenucleic acid selectively hybridizes to DNA from Salmonella typhymuriumin the presence of DNA from Klebsiella or Escherichia.
 9. An isolatednucleic acid that comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, orSEQ ID NO:18 and that can selectively hybridize to DNA from Klebsiellaoxytoca.
 10. The isolated nucleic acid of claim 9 wherein the nucleicacid selectively hybridizes to DNA from Klebsiella oxytoca in thepresence of DNA from at least one other bacterial species of the familyEnterobacteriaceae.
 11. The isolated nucleic acid of claim 9 wherein thenucleic acid selectively hybridizes to DNA from Klebsiella oxytoca inthe presence of DNA from Salmonella or Escherichia.
 12. A biosensor chipthat comprises a nucleic acid comprising SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
 13. Amethod of detecting the presence of enteric bacteria in a test samplethat comprises contacting the test sample with a probe under stringenthybridizations conditions, and detecting hybridization between the probeand a nucleic acid in the test sample, wherein the probe comprises SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, or SEQ ID NO:18.
 14. The method of claim 13 wherein the entericbacteria are of the family Enterobacteriaceae.
 15. The method of claim13 that further comprises DNA amplification.
 16. The method of claim 15wherein the DNA amplification is by polymerase chain reaction.
 17. Amethod of detecting the presence of any species of enteric bacteria in atest sample that comprises contacting the test sample with a probe understringent hybridizations conditions, and detecting hybridization betweenthe probe and a nucleic acid in the test sample, wherein the probecomprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6.
 18. The method of claim 17 wherein the enteric bacteria are of thefamily Enterobacteriaceae.
 19. The method of claim 17 that furthercomprises DNA amplification.
 20. The method of claim 19 wherein the DNAamplification is by polymerase chain reaction.
 21. A method of detectingthe presence of Escherichia in a test sample that comprises contactingthe test sample with a probe under stringent hybridizations conditions,and detecting hybridization between the probe and a nucleic acid in thetest sample, wherein the probe comprises isolated nucleic acid thatcomprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. 22.The method of claim 21 wherein the probe selectively hybridizes to DNAfrom Escherichia coli in the presence of DNA from Klebsiella,Salmonella, Shigella or Yersinia.
 23. The method of claim 21 thatfurther comprises DNA amplification.
 24. The method of claim 23 whereinthe DNA amplification is by polymerase chain reaction.
 25. A method ofdetecting the presence of Salmonella in a test sample that comprisescontacting the test sample with a probe under stringent hybridizationsconditions, and detecting hybridization between the probe and a nucleicacid in the test sample, wherein the probe comprises isolated nucleicacid that comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:14.
 26. The method of claim 25 wherein the probe selectivelyhybridizes to DNA from Salmonella typhymurium. in the presence of DNAfrom Klebsiella or Escherichia.
 27. The method of claim 25 that furthercomprises DNA amplification.
 28. The method of claim 27 wherein the DNAamplification is by polymerase chain reaction.
 29. A method of detectingthe presence of Klebsiella in a test sample that comprises contactingthe test sample with a probe under stringent hybridizations conditions,and detecting hybridization between the probe and a nucleic acid in thetest sample, wherein the probe comprises isolated nucleic acid thatcomprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. 30.The method of claim 29 wherein the probe selectively hybridizes to DNAfrom Klebsiella oxytoca in the presence of DNA from Salmonella orEscherichia.
 31. The method of claim 29 that further comprises DNAamplification.
 32. The method of claim 31 wherein the DNA amplificationis by polymerase chain reaction.
 33. A method for detecting entericbacteria in a test sample that comprises contacting a test sample with abiosensor chip that comprises a solid support and an antibody that canbind to dGTPase from Enterobacteriaceae; and detecting whether dGTPaseis bound to the biosensor chip; wherein the antibody is directed againsta peptide having SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.
 34. An isolated antibodythat can selectively bind to dGTPase from Enterobacteriaceae wherein theantibody is directed against a polypeptide having SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ IDNO:36.
 35. A method for detecting Enterobacteriaceae in a test samplethat comprises contacting the isolated antibody of claim 34 with thetest sample for a time and under conditions sufficient for the antibodyto bind to a dGTPase polypeptide so as to form a binary complex betweenat least a portion of the antibody and a portion of the dGTPasepolypeptide and detecting the binary complex.
 36. A method of isolatinga dGTPase polypeptide from Enterobacteriaceae comprising contacting asample that may contain a dGTPase from Enterobacteriaceae with theantibody of claim 34 that is attached to a solid support, washing thesolid support and eluting a dGTPase polypeptide from Enterobacteriaceae.37. A biosensor chip that comprises a solid support and an antibody thatcan selectively bind to dGTPase from Enterobacteriaceae.
 38. Thebiosensor chip of claim 36 wherein the antibody is directed against apolypeptide having SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.
 39. A biosensor chipthat comprises a solid support and a nucleic acid probe that canselectively hybridize to nucleic acid encoding a dGTPase fromEnterobacteriaceae.
 40. The biosensor chip of claim 38 wherein the probeis a nucleic acid comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.